Cells and Virus
Vero (African green monkey kidney) clone E6 cells (BEI Resources; Product No. NR-596; Manassas, VA, USA) were used to propagate SARS-CoV-2 strain New York-PV009158/2020 (NY Strain; BEI Resources; Product No. NR-53516; Manassas, VA, USA) and perform virus infectivity assays. Vero clone E6 cells were incubated at 37°C with 5% CO2 in complete Gibco Eagle’s Minimal Essential Medium (EMEM; Corning; Cat. No. 10-010-CM; Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher Scientific; Cat. No. 10082147; Waltham, MA, USA) and 100 units (U)/mL penicillin-streptomycin (PS) (ThermoFisher Scientific; Cat. No. 15140122). For SARS-CoV-2 propagation, culture media was removed from the roller bottle, 2 mL of laboratory-prepared virus stock was added (multiplicity of infection of 0.001), along with 5 mL of EMEM supplemented with 5% FBS and 100 U/mL PS, and allowed to adsorb for 1 hour at 37°C. After adsorption, 25 mL of complete EMEM (5% FBS, 100 U/mL PS) was added, and culture continued for 36 to 48 hours at 37°C. Virus was harvested when cytopathic effects (CPE) were visually apparent throughout the roller bottle and any remaining cells were removed by scraping. Infected cells were further disrupted by vortexing (ThermoFisher Scientific, Cat. No. 02215452) for 2 minutes at maximum speed and ambient temperature with sterile glass beads (Sigma-Aldrich; Cat. No. CLS72685; St. Louis, MO, USA) at a ratio of 1:7 beads to cells, then centrifuged at 800 x g for 5 minutes at 2 to 8°C to remove any remaining cellular debris. The resulting suspension was aliquoted and frozen at -80°C in single-use vials for testing. All work with SARS-CoV-2 was performed in a biosafety level-3 (BSL-3) laboratory.
Virus infectivity was quantified by endpoint dilution assay to determine the median tissue culture infectious dose (TCID50). Serial dilutions (1:10) in complete EMEM (5% FBS, 100 U/mL PS) were plated onto 80 to 90% confluent Vero E6 monolayers, with wells scored for CPE 72 hours post-infection. Quantification of the titer was determined via the Spearman-Karber method (Hamilton et al., 1977). The limit of quantitation for the SARS-CoV-2 TCID50 assay was 13.1 TCID50 (1.12 log10 TCID50) as determined by CPE observed in the lowest dilution.
Food Materials
All food materials were sourced at local grocery stores (Kroger, Meijer, and Walmart; Columbus, OH, USA). Great-Value brand uncooked, tail-on, and head-off medium shrimp with shells on were procured from Walmart. Meijer-brand full tilapia filets were used for creating coupon samples. Freshpet Select Grain Free Chicken Recipe Slice & Serve Roll Dog Food was purchased from Kroger.
Simulated Saliva
Simulated saliva was prepared in 500-mL batches as described previously (Biryukov et al., 2020). Briefly, the salts were weighed and dissolved in double deionized water, after which porcine mucin (Lee Biosolutions, Inc.; Cat. No. 435 − 11; Maryland Heights, MO, USA,) was added to a final concentration of 3 g l-1 and the pH adjusted to 7.0. Simulated saliva was stored at 2 to 8°C until use and any unused portion was discarded after one week and a new preparation made. Virus was concentrated and resuspended in simulated saliva to a final concentration of 1.3 x 105 TCID ml-1 by using a centrifugal concentrator (Spin-X UF Concentrator; Corning; Cat. No. CLS431491) with a 100-kiloDalton (kDa) molecular weight cutoff that retained the SARS-CoV-2, but allowed the complete cell culture media components to be removed.
Virus Persistence Assays
Coupons (2-cm x 2-cm) were cut from the tilapia filets and wet dog food. For shrimp, the entirety of the material served as the coupon, however, only the outside surface on the shell of the shrimp samples was used for testing. Coupons were inoculated with 100 µL of SARS-CoV-2 containing 1.3 x 104 TCID50 in simulated saliva by placing 10 droplets (10 µL each) onto each coupon, allowing the droplets to dry at ambient conditions (approximately 1 hour), and then incubating them in environmental chambers at the specified temperature (4°C or -20°C) and RH (65% or ambient). Temperature and RH were monitored with data loggers (Onset; HOBO MX1101; Bourne, MA, USA). To maintain desired humidity levels during handling, saturated salts were placed in small capacity containers (Lock-N-Lock; Seoul, Republic of Korea) to quickly return to the desired RH upon opening and closing. Positive control coupons were made identical to the test samples in triplicate and processed at T = 0, post-drying to determine the starting titer of virus inoculated on the coupons. Test coupons, in triplicate, were subjected to the specified temperature and RH with samples being taken at 6, 24, 48, 72, and 168 hours. A no-virus negative control prepared identically to the test samples was included at each time point to confirm that the food material itself was not affecting the viability assay. Virus was extracted from each coupon by placing the coupon in 10 mL of complete EMEM with 2% FBS and agitating for 15 minutes at 200 rotations per minute on a platform orbital shaker. The solution was then removed from the tube and concentrated down to 2 mL using a centrifugal concentrator (Spin-X UF Concentrator) with a 100-kDa cutoff. The 2-mL concentrated samples were filter-sterilized (ThermoFisher Scientific; Cat. No. 720–1320) through a low-binding, 0.2-µm filter before being assayed for infectious virus by the TCID50 assay, as previously described.
Persistence of Virus Genomic Material
A portion of the final extract described in the virus persistence assays was removed from the sample and stored at -80°C until virus genomic assays could be conducted. For genomic material quantification, samples were removed from frozen storage, thawed, and the RNA extracted using the IndiSpin Pathogen 96 QIAcube HT kit (INDICAL; Cat. No. SP54161; San Francisco, CA, USA) on the QIAcube HT system (QIAGEN, Inc.; Germantown, MD, USA). An approximate 0.2-mL liquid volume was used per sample. In each batch of samples extracted, an isolation positive (IP) control containing the N1 gene target and an isolation negative (IN) control not containing the N1 gene target were included as process controls and treated as all other test samples. Samples were first lysed in a buffer mixture comprised of Buffer VXL (QIAGEN; Cat. No. 1069974), proteinase K, and carrier RNA provided in the kit. The Buffer VXL mixture (0.18 mL) was added to each sample, vortexed, and then incubated at 70 ± 5°C for 10 to 15 minutes with constant agitation. Lysed samples were then transferred (approximately 0.38 mL) to the QIAcube sample block (S-block) for the automated portion of the extraction procedure. A custom QIAcube HT protocol was used to set up the QIAcube Prep Manager software, which was provided by QIAGEN to include off-board lysis and the optional DNase digestion step. Samples were loaded in the S-block to the QIAcube worktable, and the protocol initiated. The DNase digestion step was excluded, and the “top elute” reagent was omitted from the protocol (i.e., a mineral oil, which is typically used to assist with uniform elution in the final steps; however, the mineral oil may carry over to the RNA sample and interfere with downstream analyses). Purified RNA was eluted in 100 µL buffer AVE (provided in the kit), and RNA samples were stored at < -70°C until real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis.
The qRT-PCR analysis was performed using a Custom TaqMan Gene Expression Assay (ThermoFisher Scientific; Cat. No. 4332079), which is a concentrated primer-probe mixture containing the forward and reverse primers and a FAM-labeled probe using sequences specific to the SARS-CoV-2 nucleocapsid protein (N1) gene target. The primers are forward 5’- GAC CCC AAA ATC AGC GAA AT-3’; reverse 5’- TCT GGT TAC TGC CAG TTG AAT CTG-3’; and probe 5’-FAM-ACC CCG CAT TAC GTT TGG ACC-NFQ-MGB-3’. A master mix was prepared using the primer-probe reagent, TaqMan Fast Virus 1-Step Master Mix (ThermoFisher Scientific; Cat. No. 4444436) and nuclease-free water (ThermoFisher Scientific; Cat. No. AM9938). The master mix was loaded onto a PCR plate (15 µL) and a no template control comprised of nuclease-free water was added in duplicate and immediately capped. The RNA test samples (including the IN and IP) were loaded in duplicate and capped, and lastly the reference standard (RS, synthetic copy of the same N1 gene target for the primer and probe set) dilution series was loaded in triplicate and capped. All samples were added using a 5-µL volume. The qualified RS was plated as an 8-point logarithmic dilution series ranging from 5.0 x 101 to 5.0 x 107 copies per reaction, which was generated with a custom, synthetic RNA material (Biosynthesis; Lewisville, TX, USA). The synthetic RNA contains the N1 amplicon targeted by the primers and probe.
The qRT-PCR reaction was completed on a QuantStudio 6 Flex Real-Time PCR System (ThermoFisher Scientific) using a Fast Run set to hold for 5 minutes at 50°C and 20 seconds at 95°C, followed by 40 cycles of 3 seconds at 95°C and 30 seconds at 60°C. Amplification of the N1 gene target was measured by fluorescence and the response was plotted over time (i.e., cycles) to generate an amplification plot. The cycle threshold cutoff was set to 0.1 with an automatic baseline, and unknown test sample cycle threshold (CT) values were generated in the QuantStudio software based on the cycle at which their amplification curve crossed the predetermined threshold setting. The CT values were plotted against the RS curve to generate a total copy per reaction value for each well.