Dose-dependent response to infection with SARS-CoV-2 in the ferret model: evidence of protection to re-challenge

Abstract

41In December 2019 an outbreak of coronavirus disease emerged in 42 Wuhan, China. The causative agent was subsequently identified and named severe 43 acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread 44 worldwide causing a pandemic. Currently there are no licensed vaccines or 45 therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in 46 development and repurposed drugs are being tested in the clinic. There is a vital need 47 for authentic COVID-19 animal models to further our understanding of pathogenesis 48 and viral spread in addition to pre-clinical evaluation of candidate interventions. 49 50Here we report a dose titration study of SARS-CoV-2 to determine the most suitable 51 infectious dose to use in the ferret model. We show that a high (5x10 6 pfu) and medium 52 (5x10 4 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral 53 RNA shedding in both groups of six challenged animals, whilst a low dose (5x10 2 pfu) 54 resulted in only one of six displaying signs of URT viral RNA replication. The URT 55 shedding lasted up to 21 days in the high dose animals with intermittent positive signal 56 from day 14. Sequential culls revealed distinct pathological signs of mild multifocal 57 bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and 58 medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 59 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were 60 observed although animals did present with a consistent post-viral fatigue lasting from 61 day 9-14 in the medium and high dose groups. After virus shedding ceased, re-62 challenged ferrets were shown to be fully protected from acute lung pathology. The 63Page 4 of 39 endpoints of URT viral RNA replication in addition to distinct lung pathology and post 64 viral fatigue were observed most consistently in the high dose group. This ferret model 65 of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of 66 patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 67 14-21 parallel observations reported in a minority of clinical cases. 68 69 70 71 Word count: 327 72 Introduction 73 74 Coronaviruses are positive sense, single stranded RNA viruses belonging to the family 75 Coronaviridae 1 . These viruses can infect a range of animals, including humans and 76 usually cause a mild respiratory infection, much like the common cold. Two highly 77 pathogenic coronaviruses have emerged in the human population in the last 20 years; 78 severe acute respiratory syndrome (SARS) CoV and middle eastern respiratory 79 syndrome (MERS) CoV. SARS-CoV infected approximately 8,000 people worldwide with 80 a case fatality rate (CFR) of 10%, while MERS-CoV has infected approximately 2,500 81 people with a CFR of 36% 2 .82 83 In December 2019 several pneumonia cases of unknown cause emerged in Wuhan, 84 Hubei, China. Deep sequencing analysis from lower respiratory tract samples from ...

Hamster and ferret experimental infection with intranasal low dose of a single strain of SARS-CoV-2

“…In our study, all hamsters tended to gain weight overtime although at different rates, in spite of local evidence of infection. Inoculated ferrets generally present even milder clinical signs of disease, independently from the challenge dose (8)(9)(10)(11), without any weight loss, as reported in our study. The marked lethargy observed in ferrets in this study was also reported by others (8)(9)(10)(11).…”

“…Antibody kinetics in ferrets and hamsters were comparable with 1/ those seen in patients with a seroconversion between D7 and D14 (22), and 2/ those seen in experimental models using hamsters and ferrets (6,(8)(9)(10)(11). In our study, the increase in antibody levels occurred faster in hamsters than in ferrets, from no detectable level at D4 to consistently high levels between D7 and D14 in hamsters and between D7 and D10 in ferrets.…”

“…The pathogenicity of SARS-CoV2 in the nasal cavity of the hamsters infected in this study involved respiratory and olfactory epithelium, including sustentacular cells (17). In ferrets, similar infection pattern has also been observed (9). In this species, nasal washes provided non-invasive evidence of local infection with consistently high level of viral RNA recovered from D2.…”

“…Four µm sections were cut and stained with haematoxylin and eosin (H&E) and examined microscopically. In addition, samples were stained using the RNAscope in situ hybridization (ISH) technique to identify the SARS-CoV-2 virus RNA as previously described (9). Briefly, tissues were pre-treated with hydrogen peroxide for 10 mins (RT), target retrieval for 30 mins (98-101°C) and protease plus for 30 mins (40°C) (Advanced Cell Diagnostics).…”

“…Understanding the pathogenesis of the SARS-CoV-2 infection is key to maximize prevention and to develop therapeutic solutions, and this is possible through susceptible animal models such as non-human primates (cynomolgus (Macacca fascicularis) and rhesus (Macacca mulatta) macaques), cats, hamsters and ferrets (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)], being the two latest species the most economic and easiest to house and handle. To our knowledge, all ferrets and hamsters experimental infection studies (9,13),] published so far, apart from two studies (9,13),] were performed with high doses of SARS-CoV-2 (ranging from 8.10 4 TCID50 to 10 5.5 TCID50 or 10 6 pfu per animal), independently of animal weight and mainly with the objective of inducing severe infections. Here, we choose to explore the effects of a lower viral infection dose, considered closer to natural and more common infection conditions with SARS-CoV-2 in humans, probably leading to mild disease symptoms, but with potential viral excretion.…”