key: cord-0927905-g4ayxs29 authors: Freuling, Conrad M.; Breithaupt, Angele; Müller, Thomas; Sehl, Julia; Balkema-Buschmann, Anne; Rissmann, Melanie; Klein, Antonia; Wylezich, Claudia; Höper, Dirk; Wernike, Kerstin; Aebischer, Andrea; Hoffmann, Donata; Friedrichs, Virginia; Dorhoi, Anca; Groschup, Martin H.; Beer, Martin; Mettenleiter, Thomas C. title: Susceptibility of raccoon dogs for experimental SARS-CoV-2 infection date: 2020-08-20 journal: bioRxiv DOI: 10.1101/2020.08.19.256800 sha: cf36add27c879d804acc14d4e475745ee5371e2e doc_id: 927905 cord_uid: g4ayxs29 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China at the end of 2019, and became pandemic. The zoonotic virus most likely originated from bats, but definite intermediate hosts have not yet been identified. Raccoon dogs (Nyctereutes procyonoides) are kept for fur production, in particular in China, and were suspected as potential intermediate host for both SARS-CoV6 and SARS-CoV2. Here we demonstrate susceptibility of raccoon dogs for SARS-CoV-2 infection after intranasal inoculation and transmission to direct contact animals. Rapid, high level virus shedding, in combination with minor clinical signs and pathohistological changes, seroconversion and absence of viral adaptation highlight the role of raccoon dogs as a potential intermediate host. The results are highly relevant for control strategies and emphasize the risk that raccoon dogs may represent a potential SARS-CoV-2 reservoir. Our results support the establishment of adequate surveillance and risk mitigation strategies for kept and wild raccoon dogs. Article Summary Line Raccoon dogs are susceptible to and efficiently transmit SARS-CoV2 and may serve as intermediate host CoV-2 prior to the experiment. All raccoon dogs had been vaccinated against distemper, adeno-75 and parvovirus (Eurican® SHP, Merial, France). Animals were kept in individual stainless-steel 76 cages (1.5m x 0.95m x 2.0m) in four separate segments at 20°C room temperature, 60-80% 77 humidity and a 12hr/12hr (35% dimming during night modus) lighting control within a fan 78 forced draught ventilation equipped BSL3** animal facility at the Friedrich-Loeffler-Institut 79 (FLI). Water was offered ad libitum. Animals were fed daily with 400 gr commercially produced 80 feed for farmed foxes and raccoon dogs (Schirmer und Partner GmbH Co KG, Germany; 81 Michael Hassel GmbH, Langenargen, Germany). The diet was supplemented with vitamins, 82 minerals and items like one-day old chickens as described before (24). The general health status 83 of all animals, feed uptake and defecation were recorded daily. The body weight and temperature 84 of all animals were measured prior to inoculation and at days 2, 4, 8, 12, 16, 21, and 28 pi. The outline of the experiments with an observation period of 28 days is depicted in 86 Figure 1 . Nine raccoon dogs (3 males, 6 females) were infected intranasally with 10 5 TCID50 87 SARS-CoV-2 2019_nCoV Muc-IMB-1. The inoculum of 2x1ml was administered to both 88 nostrils using a pipette. To test viral transmission by direct contact, three naïve sentinel animals 89 (all female) were added 24 hours post inoculation. Nasal, oropharyngeal and rectal swabs were 90 taken at 2, 4, 8, 12, 16, 21 and 28 days post infection (dpi), blood was taken at 4, 8, 12, 16, 21 91 and 28 dpi. Two animals each were sacrificed at day 0 (control #1, #2) day 4 (animals #1, #2), 92 day 8 (animals #3, #4) and day 12 pi (animals #5, #6). The remaining inoculated animals 93 (animals #7-9) and the contacts (animals #10-12) were euthanized 28 dpi. All animals were 94 subjected to autopsy for macroscopic evaluation and tissue sampling. The animal experiments were evaluated and approved by the ethics committee of the State Office The virus was harvested after 72 h, titrated on Vero E6 cells and stored at -80°C until further use. Total RNA from nasal, oropharyngeal and rectal swab samples, fecal samples as well as 109 from tissues taken at autopsy were extracted using the NucleoMagVet kit (Macherey&Nagel, For comparison, sera were also tested in a newly developed commercial SARS-CoV-2 sVNT (28). Inoculation led to productive infection in six out of nine exposed animals. Based on the 173 lack of viral RNA detection throughout the observation period of 28 days, we concluded that 174 infection of animals #4, #8 and #9 failed (Figure 1, panel B) . While several animals showed 175 reduced overall activity at 4 dpi (animal #4, #5, #10), none of the exposed and contact animals 176 showed any obvious clinical sign of infection until the termination of the experiment. In particular, 177 neither increase in body temperature nor weight loss were observed. The serological results suggest that the induction of SARS-CoV-2 specific VNA in raccoon 276 dogs is reduced compared to ferrets but comparable to Egyptian fruit bats (23). 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