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

In December 2019 an outbreak of coronavirus disease (COVID-19) emerged in Wuhan, China. The causative agent was subsequently identified and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread worldwide causing a pandemic. Currently there are no licensed vaccines or therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in development and repurposed drugs are being tested in the clinic. There is a vital need for authentic COVID-19 animal models to further our understanding of pathogenesis and viral spread in addition to pre-clinical evaluation of candidate interventions. Here we report a dose titration study of SARS-CoV-2 to determine the most suitable infectious dose to use in the ferret model. We show that a high (5×106 pfu) and medium (5×104 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral RNA shedding in both groups of six challenged animals, whilst a low dose (5×102 pfu) resulted in only one of six displaying signs of URT viral RNA replication. The URT shedding lasted up to 21 days in the high dose animals with intermittent positive signal from day 14. Sequential culls revealed distinct pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were observed although animals did present with a consistent post-viral fatigue lasting from day 9-14 in the medium and high dose groups. After virus shedding ceased, re-challenged ferrets were shown to be fully protected from acute lung pathology. The endpoints of URT viral RNA replication in addition to distinct lung pathology and post viral fatigue were observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 14-21 parallel observations reported in a minority of clinical cases.

Study Design. Ferrets were challenged intranasally with 1ml of Victoria/1/2020 26 119 SARS-CoV-2 at three different titres representing a high, medium and low dose (Table   120 1). A high titre stock of challenge virus was prepared (passage 3), and quality control 121 sequencing showed it was identical to the original stock received from the Doherty 122 Institute and did not contain a commonly reported 8 amino acid deletion in the furin 123 cleavage site 27 . Following the initial challenge, a re-challenge with the high dose 124 (5x10 6 PFU) took place at day 28 post challenge (pc). The four (two per group) 125 remaining ferrets in groups 2 and 3 were re-challenged via the same, intranasal, route 126 and 1ml volume alongside a control group of two naïve control ferrets (group 5).
127 128 Viral Shedding following challenge. Viral RNA was detected in the nasal wash of 129 6/6 ferrets in the high dose group from day 1 pc and continued to be detected at 130 varying levels until day 20 pc (Fig. 1a). The peak in viral RNA shedding was seen 131 between day 2 and 4 pc for all ferrets in the high dose group. Following a decline in 132 viral RNA (2/2 animals) to below the limit of quantification at day 13 pc, an increase 133 was seen at days 16 and 18, with a measurement in viral RNA for one ferret (4.75x10 4 134 copies/ml) just above the limit of quantification at day 16 pc and a viral load of 1.1x10 6 135 copies/ml in the other ferret at day 18 pc. Both Group 1 survivors were euthanised on 136 day 21 at which point no viral RNA was detected in their nasal washes. In the medium dose group 6/6 ferrets also had detectable viral RNA in nasal washes 139 from day 1 pc. The peak of viral RNA shedding was more variable in the medium dose 140 group, with some ferrets peaking at days 2 to 3 pc (4/6) and others peaking at days 5 141 to 6 pc (2/6). A decline was then seen until day 11 pc where viral RNA levels fell below 142 the limit of quantification, but viral RNA was still detected. By day 16 no more viral 143 RNA was detected. Quantifiable viral RNA was only found in the nasal wash of 1/6 144 ferrets in the low challenge dose group. This ferret was euthanised on day 5 pc. No 145 other ferrets in the low dose group were found to shed quantifiable viral RNA in their 146 nasal wash.

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A similar trend in the titre of viral RNA detected in nasal wash samples was observed 149 in the throat swabs samples during the first week after challenge (Fig. 1b). The  Detection of viral RNA in the rectal swabs was found to be variable across the different 156 dose groups (Fig 1c.). The highest viral RNA load was observed in a ferret in the high 157 dose group but there was a less consistent pattern of RNA detection which did not 158 continue past day 7 pc. In the medium dose group, 4/6 ferrets were found to have 159 detectable viral RNA in their rectal swabs between day 2 and 8 pc. No viral RNA was 160 detected in any of the rectal swabs collected from the low dose group following 161 challenge.

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Page 9 of 39 163 Viral RNA was detected at quantifiable levels in the bronchoalveolar lavage (BAL) of 164 each ferret euthanised (scheduled) on day 3 pc from the high dose (1/1) and medium 165 dose (1/1) groups (Fig. 1d). Viral RNA was detected but not quantified for ferrets 166 across all three challenge groups at day 5 and 7 pc. There was no viral RNA detected    Table 2. At day 9 pc 179 all 3/3 ferrets in the high dose group showed reduced activity, a similar observation 180 was made in the medium dose group but later on day 10 pc. Reduced activity was 181 accompanied by ruffled fur, a sign that the ferrets were not grooming regularly. By         Table 3. At day 28 pc, these ferrets and two naïve control 253 animals were challenged intranasally with the high dose of SARS-Cov-2 (5x10 6 pfu). to stay above quantifiable levels in the naïve control group, although they began to fall 259 at day 8 post re-challenge (Fig. 4a). Similar results were seen in the throat swab and 260 rectal swabs (data not shown), with reduced viral shedding seen in the re-challenged 261 animals. Animals in the re-challenged medium and low dose groups exhibited weight 262 loss from baseline that was not seen at initial challenge for any of the animals in any 263 of the challenge groups (Fig. 4b). Re-challenged animals also experienced increased 264 clinical observations of lethargy and ruffled fur that was not observed at such an early 265 stage in the initial challenge ( Table 2). In contrast, the two previously naïve control 266 animals did not experience weight loss below baseline after infection and they did not 267 suffer the same level of clinical observation as the re-challenged animals (Fig 4b). the 're-challenge' (Fig. 4f). This parallels the absence of pathology observed in the 281 two naïve sentinel ferrets euthanised at day 20 pc.       showed that the response to the virus appears to be higher on re-challenge.    On day 28 pc the remaining ferrets in the low (n=2) and medium (n=2) groups were 441 re-challenged with 5x10 6 pfu by the intranasal route. Additional naïve control ferrets 442 (n=2) were also challenged on day 28, to provide a re-challenge control. All 6 animals 443 were monitored for clinical signs and one ferret from each group was euthanised on 444 day 33 and the remaining animals were euthanised on day 36.  Throat and rectal swabs were processed, and aliquots stored in viral transport media 457 (VTM) and AVL at -80C until assay.   CoV-2 NC_045512.2, with quantification between 1 x 10 1 and 1 x 10 7 copies/µl.

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Positive samples detected below the limit of quantification were assigned the value of 505 6 copies/µl, whilst undetected samples were assigned the value of ≤ 2 copies/µl, 506 equivalent to the assays limit of detection.