Susceptibility of domestic swine to experimental infection with SARS-CoV-2

SARS-CoV-2, the agent responsible for COVID-19 has been shown to infect a number of species. The role of domestic livestock and the risk associated for humans in close contact remains unknown for many production animals. Determination of the susceptibility of pigs to SARS-CoV-2 is critical towards a One Health approach to manage the potential risk of zoonotic transmission. Here, pigs undergoing experimental inoculation are susceptible to SARS-CoV-2 at low levels. Viral RNA was detected in group oral fluids and nasal wash from at least two animals while live virus was isolated from a pig. Further, antibodies could be detected in two animals at 11 and 13 days post infection, while oral fluid samples at 6 days post inoculation indicated the presence of secreted antibodies. These data highlight the need for additional livestock assessment to better determine the potential role domestic animals may contribute towards the SARS-CoV-2 pandemic.


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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent of 30 coronavirus disease  was recently identified to cause severe respiratory distress in 31 humans with symptoms ranging from asymptomatic, mild to severe, and sometimes fatal cases 32 (1). Rapidly spreading, this novel virus emerged in Wuhan China, to generate a pandemic as 33 declared by the World Health Organization on March, 11 th 2020 (2). Predicted to have 34 originated in bats, SARS-CoV-2 origins are still under intense investigation as reports continue 35 to identify the ability of the virus to infect new animal species (3)(4)(5)(6)(7)(8). Detection of natural 36 infections has recently shed light on knowledge gaps in understanding transmission which has 37 raised concerns regarding amplifying or reservoir hosts. In turn, a better understanding of 38 wildlife and domestic animal susceptibility is required to assess the potential roles and present 39 risks to prevent future spread of disease. Domestic swine, one of the most significant and highly 40 produced agricultural species with previous impacts to public health, must be assessed (9-12).

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The increase in "backyard" small stakeholder animal production in both rural and urban 42 environments provides an important source of high-quality protein and income, but can also 43 serve as a source for zoonotic disease; therefore, it is important to investigate their potential role 44 during SARS-CoV-2 spread (13). Evidence for the involvement of production animals was 45 recently highlighted in The Netherlands where anthroponotic transmission of SARS-CoV-2 from 46 humans to farmed mink with subsequent zoonotic transmission to at least two humans from mink 47 has been proposed, further exemplifying the need to identify the potential role of production 48 animals in disease transmission (14). 49 Angiotensin-converting enzyme 2 (ACE2) has been identified to be the receptor for 50 SARS-CoV-2 (15). A Basic Local Alignment Search Tool (BLAST) query of the protein 51 database using translated nucleotide (BLASTx) from the human ACE2 coding sequence predicts 52 98% coverage and 81% identity for the homologous receptor in swine. Interestingly, using the 53 same search both mink (82%) and feline (85%) show similar identity to the human ACE2 for 54 their cognate receptors. Moreover, both mink and cats have been reported to be susceptible to 55 SARS-CoV-2 and have shown transmission to other animals (5, 16). Work by Zhou et al. 56 utilized in vitro infectivity studies testing ACE2 receptor from laboratory mice, horseshoe bats, 57 civets and the domestic pig. All of the respective receptors, except mice, were reported to enter 58 HeLa cells indicating a functional target for SARS-CoV-2. Moreover, the authors employed 59 additional known coronavirus receptors including both aminopeptidase N and dipeptidyl 60 peptidase 4 finding neither are used for cell entry outlining the specificity for the ACE2 receptor 61 (17).

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The work reported here aims to determine whether domestic swine are susceptible to 63 SARS-CoV-2 infection, providing critical information to aid public health risk assessments.

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Following oronasal inoculation, swine were assessed for: clinical signs and pathology, evidence 65 of virus shedding, viral dissemination within tissues, and seroconversion. The data presented in 66 this study provides evidence live SARS-CoV-2 virus can persist in swine for at least 13 days 67 following experimental inoculation. guidelines. Group housing was carried out in the BSL-3zoonotic large animal cubicles, and 75 animals were provided with commercial toys for enrichment and access to food and water ad 76 libitum. All invasive procedures, including experimental inoculation and sample collection (nasal 77 washes, rectal swabs, and blood collection) were performed under isoflurane gas anesthesia, and    Hematology, chemistry, and blood gas analyses. Hematology was performed on an HM5 105 analyzer (Abaxis) using K3 EDTA-treated whole blood and the following parameters were 106 evaluated: red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular 107 hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution weight, platelets, 108 mean platelet volume, white blood cells, neutrophil count (absolute (abs) and %), lymphocyte 109 count (abs and %), monocyte count (abs and %), eosinophil count (abs and %), and basophil 110 count (abs and %). Blood chemistries were evaluated on a VetScan 2 (Abaxis) with the 111 Comprehensive Diagnostic Profile rotor (Abaxis) using serum stored at -80˚C until tested and the 112 following parameters were evaluated: glucose, blood urea nitrogen, creatinine, calcium, albumin, 113 total protein, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, 114 amylase, potassium, sodium, phosphate, chloride, globulin, and total bilirubin. Sodium heparin 115 treated blood was used to analyze venous blood gases, which were performed on an iSTAT 116 Alinity V machine (Abaxis) using a CG4+ cartridge (Abaxis) to measure the following 117 parameters: lactate, pH, total carbon dioxide, partial pressure carbon dioxide, partial pressure

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Experimental inoculation of sixteen eight week old swine was performed oronasally with 220 1 X 10 6 pfu of SARS-CoV-2, distributed evenly between both nostrils and the distal pharynx.

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Starting at 1 day post inoculation (DPI), pigs developed a mild, bilateral ocular discharge and in 222 some cases, this was accompanied by serous nasal secretion. This was observed for only the first 223 three days post inoculation. Temperatures remained normal throughout the study (Table S1).

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Overall, animals did not develop clinically observable respiratory distress, however one animal 225 (Pig 20-06) presented mild depression at 1 DPI accompanied with a cough which was maintained 226 through 4 DPI. This animal did not display additional clinical signs over the course of the study.  (Table 1).

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Every other day starting at 3 DPI to 15 DPI, oral, nasal, and rectal swabs were sampled to 232 evaluate the potential for delayed onset (1). Nucleic acid was extracted from swabs and RT-233 qPCR was performed to identify SARS-CoV-2 by targeting the envelope gene (E gene). Viral 234 RNA could not be detected in swabs from any animals over the course of the study (Table 2, A).

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Nasal washes are a sensitive method for detection of pathogens in swine and were routinely 236 sampled using sterile D-PBS to rinse nasal passages. Two pigs (20-10, 20-11) displayed low 237 levels of viral RNA by RT-qPCR at 3 DPI (Table 2,   Detection of SARS-CoV-2 was also attempted from whole blood by RT-qPCR, following 253 the sampling schedule outlined in Table 1. As outlined in Table 2A, viremia, as indicated by the 254 presence of viral RNA in the blood, could not be detected in any animal throughout the study.

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Blood cell counts, chemistries, and gasses were measured using the Abaxis HM5, VetScan 2, and 256 iSTAT respectively. Although some variation was observed throughout the study, changes were 257 minimal and inconclusive, and profiles consistent with acute viral infection or subsequent organ 258 damage were not observed.

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To identify potential target tissues or gross lesions consistent with SARS-CoV-2 disease, 261 necropsy was performed on two animals starting at 3 DPI and every other day up to day 15; with 262 an additional two pigs necropsied at both 22 and 29 DPI (Table 1) (Table 2).

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The development of SARS-CoV-2 neutralizing antibodies were monitored over the 280 course of study. Starting at 7 DPI, serum was obtained from individual animals for both virus 281 neutralization test (traditional VNT) and a surrogate virus neutralization test (sVNT; Genscript).

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Sera was first tested using a traditional VNT, with one pig (20-07) generating neutralizing 283 antibody titers, albeit weak, at a 1:5 dilution with a 70% reduction of plaques at both 13 and 15 284 DPI (Table 3). Consequently, the sVNT assay identified the same animal, Pig 20-07, as antibody 285 positive with 0.188 µg/ml antibody at 15 DPI. A second pig (20-14) was shown to have 286 generated antibody at 11 DPI (0.113 µg/ml) and 13 DPI (0.224 µg/ml). The sVNT was also 287 employed to identify secreted antibody in oral fluids throughout the study. Interestingly, at 6 DPI 288 we detected positive antibody (0.133 µg/ml) from group oral fluid collected from cubicle 1 289 (Table 3). The results presented in this study define domestic swine as a susceptible species albeit at 293 low levels to SARS-CoV-2 viral infection. One animal was found to retain live virus, while two 294 additional animals had detectible RNA measured in the nasal wash, and two pigs developed 295 antibodies. In total, of the sixteen animals experimentally inoculated, five displayed some level We would like to thank the Public Health Agency of Canada for SARS-CoV-2 isolate for 330 this study, in addition the Animal Care and Genomics units for their support during this project. 331 We would also like to thank Dr. Claire Andreasen for her review of the clinical pathology