Virus and cells
Vero E6 cells (ATCC® CRL-1586™; American Type Culture Collection, Manassas, VA, USA) were used for the cultivation and titration of SARS-CoV-2. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, New York, NY USA), supplemented with 5% fetal bovine serum (R&D Systems, Minneapolis, MN, USA) and antibiotics/antimycotics (Thermo Fisher Scientific, Waltham, MA, USA), called DMEM growth medium, at 37 °C under a 5% CO2 atmosphere in a cell culture incubator. The SARS-CoV-2 USA-WA1/2020 strain was acquired from the Biodefense and Emerging Infection Research Resources Repository (BEI Resources, Manassas, VA, USA) and was passaged three times on Vero E6 cells with a final titer of 7 × 105 50% tissue culture infective dose (TCID50)/ml.
Preparation of test substrates
The stability of SARS-CoV-2 was tested in the following house fly feeding substrates: egg yolk powder (Bulkfoods.com, Toledo, OH, USA), non-fat milk powder (GreatValue; Walmart Inc., Bentonville, AR, USA) and powdered sugar (GreatValue, Walmart). A 10% (w/v) virus-spiked substrate was prepared by mixing each substrate with virus in DMEM growth medium. Additionally, a virus-spiked medium (positive control) was prepared by adding virus to DMEM growth medium. All virus-spiked substrates and the positive medium control contained a final SARS-CoV-2 concentration of 5 × 104 TCID50/ml. DMEM growth medium with no virus served as the negative control.
Stability of SARS-CoV-2 in various substrates
Ten individual cotton wicks (approx. 0.25 cm3 in size) were placed into separate wells of four different 12-well tissue culture plates (Corning). Two ml of three different substrates were added to six wicks and 2 ml of control substrate was added to four wicks. The final concentration of virus was 105 TCID50 per well. The plates were incubated in an environmental chamber at 22 °C and 60% relative humidity. The substrates were collected by adding 1 ml of DMEM growth medium onto the wicks and then, using a micropipettor, the medium was pipetted up and down 5 times before collection. The samples were collected at 0, 1, 4 and 24 h post-inoculation from the plates and stored at − 80 °C until analyzed. The virus titer of each sample was determined by performing the TCID50-cytopathic effect (CPE) assay (see below).
House fly maintenance
House flies (Musca domestica) were maintained at the U.S. Department of Agriculture, Center for Grain and Animal Health Research, Arthropod-Borne Animal Diseases Research Unit (ABADRU-USDA). Female house flies (1–2 days old, unmated) were fasted and water was removed for 12–16 h prior to exposure to the feeding substrate. On the day of the study, flies (n = 60) were anesthetized with CO2 for 1–2 s and placed into 12 individual polypropylene containers (n = 5/container), approximately 250 ml in size. Each container contained two cotton wicks placed in a small weigh boat and the container was sealed with a mesh lid to prevent fly escape while allowing air circulation. Primary containers were placed in a secondary container and were transported to the Biosecurity Research Institute (BRI) at Kansas State University for studies under Arthropod Containment Level-3 (ACL-3) conditions.
Virus acquisition by house flies
House flies were anesthetized by exposure to cold (− 20 °C for 5 min), then one of the three feeding substrates was introduced to four fly containers each by pipetting 2 ml onto the cotton wicks. The feeding substrates were: (i) SARS-CoV-2 in 10% non-fat milk feeding substrate; (ii) SARS-CoV-2 in DMEM growth medium (positive control); or (iii) DMEM growth medium without virus (negative control). The final concentration of SARS-CoV-2 in the spiked feeding substrate and in the positive control was 1 × 105 TCID50. At 4 h post-exposure, two containers from each of the three groups were placed at − 20 °C for 30 min to sacrifice the flies. Then, individual flies were collected and placed in tubes with 1 ml transport medium (199E medium, 200U/ml penicillin, 200 µg/ml streptomycin, 100 µg/ml gentamycin and neomycin, 5 µg/ml amphotericin B; Sigma-Aldrich, St. Louis, MO, USA). Transport medium was used for storage and as a processing medium for the collected samples. Container swabs were collected by wiping the interior surface of the containers with a sterile cotton swab soaked in 1 ml of transport medium; they were re-immersed into the same transport medium for further evaluation. This procedure was repeated on the remaining two containers from each group after 24 h exposure (Fig. 1). The collected flies and container swabs were stored at − 80 °C until further processing for infectious virus and viral RNA.
Virus transmission by house flies
A second set of house flies (n = 60) were fed with the three feeding substrates as described above for a 24 - time period. Exposed flies were then transferred to a new set of 12 containers containing 2 ml of virus-negative test substrate, i.e. 10% non-fat milk powder in growth medium on cotton wicks. The flies were kept for 4 or 24 h post-transfer. At each time point, the flies, the substrate and the inner surface of the containers were swabbed and collected as described above and the swabs and flies stored at − 80 °C until further processing.
Sample processing
Each fly collected during the experiments described above was homogenized using a Tissuelyser II (Qiagen, Germantown, MD, USA). Briefly, individual flies were placed in a 1.5-ml microcentrifuge tube containing one sterile 3-mm tungsten carbide bead (Qiagen) and 1 ml of virus transport medium. The flies were homogenized for 30 cycles for 3 min. Homogenates were filtered through a 0.22-µm polyethersulfone (PES) membrane filter (MIDSCI, St. Louis, MO, USA). The collected swabs were removed from the tubes with the transport medium and then filtered as descibed above.
RNA extraction and quantitative reverse transcription-PCR
RNA was extracted from 50 µl of filtered house fly homogenate, 140 µl of filtered swab medium and 140 µl of the respective feeding substrate solution using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The quantitative reverse transcription-PCR (RT-qPCR) assay was performed according to the RT-qPCR protocol established by the US Centers for Disease Control and Prevention (CDC) for the detection of the SARS-CoV-2 nucleocapsid (N)-specific RNA (https://www.fda.gov/media/134922/download) using the N2 primer and probe set and the qScript XLT One-Step RT-qPCR Tough Mix (Quanta Biosciences, Beverly, MA, USA) on a CFX96 real-time thermocycler (Bio-Rad, Hercules, CA, USA). The PCR plate controls included a quantitated SARS-CoV-2 N-specific qPCR positive control, diluted 1:10 (Integrated DNA Technologies, Coralville, IA, USA), and a non-template control (NTC). The results were analyzed using Bio-Rad CFX Manager 3.1 software. Samples with Cq values of < 38 were considered positive for SARS-CoV-2 RNA. A reference standard curve method using SARS-CoV-2-specific RNA as a standard was used for calculating the SARS-CoV-2 copy number of each sample, as previously described [3, 19].
Virus isolation
Vero E6 cells were plated at a density of 1 × 104 cells in 100 µl medium per well in 96-well plates. After overnight incubation, 50 µl of filtered fly homogenates or 50 µl of filtered swab solution was pipetted into each well in duplicate. Feeding substrates were diluted 1:10 in transport medium and filtered as described above, then 50 µl was pipetted into duplicate wells. Every sample was blind passaged three times at 3 days post-infection (dpi) by transferring approximately 150 µl of the culture medium from each well onto a new Vero E6 monolayer in 96-well plates.
Immunofluorescent assay
After the first and third passage, Vero E6 monolayers were fixed and stained with mouse monoclonal antibodies specific for the SARS-CoV-2 RBD region and a fluorescein-labeled goat anti-mouse antibody as previously described [11]. Monolayers were examined for fluorescein isothiocyanate-positive cells with an EVOS fluorescent microscope (Thermo Fischer Scientific, Waltham, MA, USA). Mock-infected and SARS-CoV-2 infected Vero E6 cells were used as negative and positive controls, respectively, as shown in Additional file 1: Figure S1.
TCID50-CPE assay
To determine virus titer, each sample was tenfold serially diluted in DMEM growth medium and added onto Vero E6 cell monolayers in 96-well plates. The inoculated wells were observed for the presence of CPE after 3–5 dpi. The virus titer of each sample was calculated using the Spearman–Karber method [21].