key: cord-0976265-xynsy7z8
authors: Francisco, Raquel; Hernandez, Sonia M.; Mead, Daniel G.; Adcock, Kayla G.; Burke, Sydney C.; Nemeth, Nicole M.; Yabsley, Michael J.
title: Experimental susceptibility of North American raccoons (Procyon lotor) and striped skunks (Mephitis mephitis) to SARS-CoV-2
date: 2021-03-08
journal: bioRxiv
DOI: 10.1101/2021.03.06.434226
sha: f023948f989eecda6a1d6c2f1ed437d91bc7b4a3
doc_id: 976265
cord_uid: xynsy7z8

Skunks and raccoons were intranasally inoculated or indirectly exposed to SARS-CoV-2. Both species are susceptible to infection; however, the lack of, and low quantity of infectious virus shed by raccoons and skunks, respectively, and lack of cage mate transmission in both species, suggest that neither species are competent SARS-CoV-2 reservoirs. Article Summary Line Experimental SARS-CoV-2 inoculation of North American raccoons and striped skunks showed susceptibility to infection, but transient, low-level shedding suggests that neither species is likely to be a competent natural reservoir.

SARS-CoV-2 continues to circulate on a global scale, the need to identify potential 21 animal reservoirs, especially among wildlife, has become a priority, spurring surveillance and susceptibility trials of numerous species (1,2). Spillback infections from humans to animals has 23 raised concerns about SARS-CoV-2 becoming endemic in native wildlife species (3). 24 The species belonging to Musteloidea (Mustelidae, Mephitidae, and Procyonidae) are 25 ecologically and taxonomically relevant due to their high probability of becoming exposed to, 26 infected with, and developing clinical disease to SARS-CoV-2 (4). For example, ferrets, close 27 relatives to North American Musteloidea, are well-established animal models for coronaviruses 28 (5) and are highly susceptible to SARS-CoV-2 (6-8). Skunks and raccoons are also notorious reservoirs of viruses that have substantial impacts on 35 other wildlife and humans (i.e., rabies, canine distemper, protoparvovirus) (11,12). We therefore 36 evaluated the susceptibility to infection, seroconversion, transmission potential between 37 conspecifics, tissue tropism and pathology associated with SARS-CoV-2 in striped skunks and 38 raccoons.

The Study 40 We performed two independent susceptibility trails, first on raccoons and then skunks, 41 using a similar experimental design. We directly inoculated 4 animals intranasally with 10 3 ("low 42 dose") and 4 with 10 5 ("high dose") PFU of SARS-CoV-2 (direct inoculation, hereafter DI), 43 strain SARS-CoV-2 USA-WA1/2020. This high dose has produced infections in ferrets and 44 other species (7,13). The low dose was used to mimic the amount of virus to which these species 45 may be naturally exposed (e.g., through consuming human garbage or potentially animal-to-46 animal) and has also resulted in infections and clinical disease in ferrets (14) . DI animals were 47 housed in pairs within ~1.5x1.5x2m wire mesh cages (Appendix Figure) . We introduced a 48 single, naïve conspecific to each pair at 48hrs after inoculation to test for direct contact 49 (hereafter, DC) transmission. We housed 4 naïve animals of each species separately as controls 50 and they were subjected to the same sampling routine as the experimental animals. Additional 51 details are located in the Technical Appendix.

To document viral shedding, we collected nasal and rectal swabs daily from days post 53 inoculation (DPI) 1-5, then on DPI 7, 9, 17 and DPI 7, 9, 15 for DI raccoons and skunks, 54 respectively. DC raccoons and skunks were swabbed on DPI 1-5 and on DPI 7, 9, 11, 17 and DPI Transcriptase PCR (rRT-PCR) that targeted both the N1 and N2 genes (15). All positive VI 58 samples were confirmed with rRT-PCR. Viral titers were quantified using plaque assays.

Shedding was detected from two high dose skunks on DPI 3, 4, and 5 and DPI 1 and 2, 60 respectively. The highest shed titer was 3.3 log10/mL on DPI 4 (Appendix Table 1 While currently it seems unlikely for SARS-CoV-2 to circulate in raccoon and skunk 108 populations and for virus to spillback into humans, other taxonomically related species, such as 109 fishers, martins, and black-footed ferrets, especially those of conservation concern, have yet to 110 be studied. Given that we did not document viral shedding, even in seroconverted raccoons, 

Animals and SARS-CoV-2: Species susceptibility and viral 132 transmission in experimental and natural conditions, and the potential implications for 133 community transmission

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Transmission of SARS-CoV-2 on mink farms between humans and 141 mink and back to humans

Could Mustelids spur COVID-19 into a panzootic? Vet 144

SARS virus infection of cats and ferrets

Infection and Rapid 150 Transmission of SARS-CoV-2 in Ferrets

Susceptibility of ferrets, cats, 153 dogs, and other domesticated animals to SARS-coronavirus 2

SARS-CoV-2 is transmitted via contact and via the air between ferrets

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Current and future climatic regions favourable for 161 a globally introduced wild carnivore, the raccoon Procyon lotor

Skunk Rabies

Infectious Diseases of Wild Mammals

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The SARS-CoV ferret model in 172 an infection-challenge study

Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome 176

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