Serologic Surveillance for SARS-CoV-2 Infection among Wild Rodents, Europe

We report results from serologic surveillance for exposure to SARS-CoV-2 among 1,237 wild rodents and small mammals across Europe. All samples were negative, with the possible exception of 1. Despite suspected potential for human-to-rodent spillover, no evidence of widespread SARS-CoV-2 circulation in rodent populations has been reported to date. Esitämme tulokset serologisesta tutkimuksesta, jossa seulottiin SARS-CoV-2 tartuntojen varalta 1,237 luonnonvaraista jyrsijää ja piennisäkästä eri puolilta Eurooppaa. Kaikki näytteet olivat negatiivisia, yhtä näytettä lukuun ottamatta. SARS-CoV-2:n läikkymisen ihmisistä jyrsijöihin on arveltu olevan mahdollista, mutta todisteet viruksen laajamittaisesta leviämisestä jyrsijäpopulaatioissa puuttuvat.

This appendix contains methodological details on sample collection (field samples and experimental controls) and the laboratory diagnostic assays (serologic and molecular). The final section consists of the work's legal and ethical statements.

Sampling Field Samples
Small mammals were trapped using snap-traps (Germany) or live traps (all countries) whose size was adapted to the target species (e.g., large wire mesh traps for rats, Sherman or Longworth traps for Myodes, Microtus, or Apodemus spp.). Traps were set either following predetermined transect lines, or at specific sites where rodents had been seen by site managers (details available upon request). Traps were left on each sampling site for one to eleven nights. Live traps were checked every morning, and re-filled with hydrophobic cotton or straw and food (seeds, carrots, sardine, peanut butter) daily to provide resources and a suitable environment for trapped animals.
Trapped animals were identified using morphological criteria in the field. When field identification was problematic, molecular identification was performed in the lab: Microtus species were identified using Sanger sequencing of CO1 fragment (1) and Apodemus sylvaticus and A. flavicollis were distinguished using the AP-PCR as in (2).
Live-trapped animals were euthanized with an overdose of isoflurane. Rodents found dead by site managers were also included in the study. All animals were dissected and several organs were collected (including the heart, placed in PBS for serologic assaying, and colon, placed in RNAlater), and stored at -20°C until assayed. Individual characteristics were also recorded (mass, body length, gender, sexual characteristics).
In total we sampled 853 animals from forests and 384 from urban parks. A breakdown of the samples collected by host species, localities and dates is provided in Appendix 2 Page 2 of 9 (https://wwwnc.cdc.gov/EID/article/28/12/22-1235-App1.xlsx). All legal and ethical information regarding this study is collated in a dedicated section at the end of this Appendix.

Vaccinated Animals
Ten-week old Syrian golden hamsters (Mesocricetus auratus) were acclimatized at the University of Helsinki biosafety level 3 (BSL-3) facility for 7 days in individually ventilated biocontainment cages (ISOcage; Scanbur) with one hamster per cage. Animals were then immunized twice 7 days apart with an experimental receptor binding domain-based nasal vaccine (patent pending). Immunized hamsters were euthanized by cervical dislocation 14 days after the second immunization and heart was collected into PBS and stored at -20°C.

Challenged Animals
Virus. The challenge SARS-CoV-2 strain used in these experiments was prepared as described previously (3)  At fourteen days post-infection, the blood was collected by heart puncture in 4 mL EDTA 3K Vacutest tubes. The plasmas were obtained after centrifugation (15 min, 1000 g) and stored at -16°C until analysis. The presence of SARS-CoV-2 neutralizing antibodies was confirmed in these samples by seroneutralization (see methods details below) before IFA testing.
Heart Samples. Six hamsters were anesthetized with isoflurane and intranasally inoculated with 40 µL containing 10 5 TCID50 of virus (20 µL in each nostril). At fifteen days post-infection, after exsanguination, the hearts were collected in vials containing 500 µL of sterile PBS. The samples were then stored at -16°C until analysis. The presence of SARS-CoV-2 antibodies in these samples was confirmed by microsphere immunoassay (see methods details below) before IFA testing. Figure S2 shows representative IFA slides, including three different positive controls and three field samples (two negative and the one positive).

Laboratory Diagnostic Procedures Immunofluorescent Assay (IFA)
All field samples were screened for SARS-CoV-2 antibodies using an immunofluorescent assay (IFA) based on the SARS-CoV-2/Finland/1/2020 virus as described in (4), with the following modifications: • Samples consisted of whole rodent hearts in PBS, whose supernatant was assayed undiluted, and The capacity of this test for robust detection of rodent SARS-CoV-2 antibody response was assessed using a range of animal experiments comparing: • different immunization methods (vaccination versus experimental infection), • different sample types (plasma versus heart in PBS), • different secondary conjugates (anti-mouse versus anti-hamster), Results were consistently positive for all tested combinations of the above factors (Appendix Table). The corresponding animal procedures are detailed in section A.2. above.

Confirmatory Serologic Assays
The IFA-positive field sample was subjected to two further SARS-CoV-2 serologic assays: a microsphere immunoassay and seroneutralization. These assays were also used to confirm the experimental positive controls described above before IFA testing.

Microsphere Immunoassay
This assay was carried out as described in (7) with the following modifications: the three recombinant SARS-CoV-2 antigens used to capture SARS-CoV-2 specific antibodies were the Nucleoprotein, the Spike Glycoprotein (S1) RBD and the Spike Glycoprotein On the following day, serum samples as well as positive and negative internal controls were serially diluted (1 in 3 dilution steps) in culture medium. Fifty microliters of culture medium containing approx. 100 TCID50 (back-titrated during the seroneutralization assays) of SARS-CoV-2 virus strain UCN19 (8) were then added to the diluted sera. The plates were incubated at 37°C with 5% CO2 for 1 h to allow neutralisation complexes to form between the neutralizing antibodies and the virus. Afterwards, the cell culture supernatants were removed and replaced with 100 µL of the virus + serially diluted sample (or control) mixes. The microplates were then incubated at 37°C in a humid chamber containing 5% CO2 for at least 3 days. Plates were then read using an "all or nothing" (binary) scoring method for the presence of viral cytopathic effect (CPE). The neutralisation titers were based on the highest dilution that prevented discernible cytopathic effect. The IFA positive sample was assessed both after a 500 g x 5 min centrifugation starting at a 1:10 dilution, and without centrifugation starting from the neat sample. It was negative in both experiments.

PCR screening of Fort 6 samples (Belgium)
The 59 animals from the Fort 6 location near Antwerp, Belgium, where the seropositive rodent had been detected, were screened for SARS-CoV-2 infection using a specific PCR. Total RNA was extracted from rodent colon samples using the QIAamp 96 Virus QIAcube HT kit (Qiagen). Colon samples were first removed from RNAlater in a BSL2 laboratory and approx. Ten mg were placed in 180 µL ATL buffer + 20 µL proteinase K (supplied with the kit) in secure 2 mL tubes containing two glass bead and autoclaved sand.
The tubes were then incubated at 56°C for 30 min for enzymatic lysis, after which they were shaken at 30 Hz for 2 × 2 min using a TissueLyser (Qiagen). Lysates were then spun at 500 g for 5 minutes, and 200 µL clear supernatant were used as starting material for automated QIAcube extraction as per manufacturer's instruction, with the following modification: the final target elution volume was 120 µL. Eluted RNA were then stored at -80°C until assayed by PCR. Tissues from SARS-CoV-2 challenged rodents were used as positive extractions controls with every extraction batch and returned consistent positive PCR.

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Extracted RNA were then assayed using the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit (New England BioLabs Inc, MA USA) as per manufacturer's instructions with no modification (using the provided kit positive control).

Belgium
The procedures were approved by the University of Antwerp Ethical Committee for Animal Experiments (permit number 2020-21). Small mammal trapping was approved by the