key: cord-0913003-kyq4h4f4
authors: Monchatre-Leroy, Elodie; Lesellier, Sandrine; Wasniewski, Marine; Picard-Meyer, Evelyne; Richomme, Céline; Boué, Franck; Lacôte, Sandra; Murri, Séverine; Pulido, Coralie; Vulin, Johann; Salguero, Francisco J; Gouilh, Meriadeg Ar; Servat, Alexandre; Marianneau, Philippe
title: Hamster and ferret experimental infection with intranasal low dose of a single strain of SARS-CoV-2
date: 2020-09-24
journal: bioRxiv
DOI: 10.1101/2020.09.24.311977
sha: 6033c4754d0918ddd55c312e9e5cdc1aad13b758
doc_id: 913003
cord_uid: kyq4h4f4

Understanding the pathogenesis of the SARS-CoV-2 infection is key to develop preventive and therapeutic strategies against COVID-19, in the case of severe illness but also when the disease is mild. The use of appropriate experimental animal models remains central in the in-vivo exploration of the physiopathology of infection and antiviral strategies. This study describes SARS-CoV-2 intra-nasal infection in ferrets and hamsters with low doses of low-passage SARS-CoV-2 clinical French isolate UCN19, describing infection levels, excretion, immune responses and pathological patterns in both animal species. Individual infection with 103 pfu SARS-CoV-2 induced a more severe disease in hamsters than in ferrets. Viral RNA was detected in the lungs of hamsters but not of ferrets and in the brain (olfactive and/or spinal bulbs) of both species. Overall, the clinical disease remained mild, with serological responses detected from 7 days and 10 days post inoculation in hamsters and ferrets respectively. Virus became undetectable and pathology resolved within 14 days. The kinetics and levels of infection can be used in ferrets and hamsters as experimental models for understanding the pathogenicity of SARS-CoV-2, and testing the protective effect of drugs.

greatest. To fight COVID-19, neither vaccines nor therapeutic treatments are currently available. Understanding 38 the pathogenesis of the SARS-CoV-2 infection is key to maximize prevention and to develop therapeutic 39 solutions, and this is possible through susceptible animal models such as non-human primates (cynomolgus 40 (Macacca fascicularis) and rhesus (Macacca mulatta) macaques), cats, hamsters and ferrets (4-13)], being the 41 two latest species the most economic and easiest to house and handle. To our knowledge, all ferrets and hamsters 42 experimental infection studies (9,13),] published so far, apart from two studies (9,13),] were performed with 43 high doses of SARS-CoV-2 (ranging from 8.10 4 TCID50 to 10 5.5 TCID50 or 10 6 pfu per animal), independently 44 of animal weight and mainly with the objective of inducing severe infections. Here, we choose to explore the 45 effects of a lower viral infection dose, considered closer to natural and more common infection conditions with 46 SARS-CoV-2 in humans, probably leading to mild disease symptoms, but with potential viral excretion.

The main objectives of the study were a) to characterize the kinetics of the disease (clinical signs, 48 pathogenicity and immune responses) in ferrets and hamsters infected with low doses of low-passage SARS-49 CoV-2 clinical isolate, in order to develop suitable animal models for therapeutic and vaccine studies.

SARS-CoV-2 viral strain UCN19 was isolated during the course of the active epidemic from naso-53 pharyngeal flocked swabs obtained from patients at the University Hospital of Caen, Normandy, France, who 54 were suffering from respiratory infection and were confirmed infected by SARS-CoV-2 by routine molecular 55 diagnosis. The swabs were eluted in UTM media (Copan, Italy) at 4°C for less than 48 hours. Vero CCL-81 56 cells (passage 32, from ATCC, USA), grown at 80% confluence level were inoculated with 200µl micro-filtered 57 elution. Cells were visually checked for cytopathic effect on a daily basis using an inverted microscope. Cell 58 supernatants (12 ml) were harvested at day 3 after inoculation and immediately used for passage 1 (P1) produced 59 in T75 culture flasks containing Vero cells as previously described. P1 was used for stock production of UCN19, 60 aliquoted and stored at -80°C before titration, genomic quantification, sequencing and experimental infections 61 to ferrets and hamsters. The use of the low-passage SARS-CoV-2 clinical isolate P1 reduces the risk of cell-62 culture induced genetic modification. 

Fifteen 10 month-old ferrets (Mustela putorius furo, ten neutered males and five females Euroferrets, 69 Denmark) and twenty-one 8-week old female hamsters (Mesocricetus auratus, strain RjHan:AURA -Janvier 70 Labs, France) were used. Ferrets and hamsters were kept in cages with environmental enrichment, allocated 71 into groups of 2 to 5 ferrets and 2 hamsters per cages, with non-infected animals kept in separate room from the 72 infected animals. Food and water were provided ad-libitum. Weight, body temperature (measured once a day 73 by subcutaneous chips IPTT300, Plexx, the Netherlands) and activity levels of all animals were monitored and 74 recorded on a daily basis throughout the duration of the experimental procedures.

Twelve ferrets and fifteen hamsters were anesthetized with isofluorane and inoculated by the intranasal 76 route with 2.10 3 pfu and 1.8.10 3 pfu of UCN19 SARS-CoV-2 strain respectively. The doses ranged from 1 to 3 77 pfu per gram of ferret (average 2 pfu/g ferret) and from 20 to 25 pfu per gram of hamster (average of 22 pfu/g 78 of hamster). Ferrets received the inoculum in 250 µl and hamsters in 20 µl in each nostril. At day 2 (D2) post-79 inoculation, D4, D7, D10 and D14, infected animals were anaesthetized with isoflurane for sample collection: 80 oro-pharyngeal swabs (plain swab rayon tipped, Copan, Italy), nasal washes in ferrets only (by administering 81 500ml PBS to each nostril) hamsters being too small for this sampling technique, rectal swabs in ferrets (plain 82 swab rayon tipped, Copan, Italy) and fecal pellets spontaneously produced in hamsters. On the same time-83 points, blood was collected from the cranial vena cava in ferrets using vacutainer (SST tubes, BD) and from the 84 retro-orbital vein in hamsters to isolate serum for immunological tests. ) 98 by plaque assay on VeroE6 cells. A 12 well-plate was seeded with a VeroE6 cell suspension 24 hours before 99 virus inoculation. Organs were harvested, weighted and homogenized in appropriate volumes of DMEM with 100 stainless steel beads (QIAGEN) for 3min at 30 Hz using TissueLyserII (QIAGEN). Homogenates were then 101 clarified by centrifugation (2,000 × g at 4°C for 10 min), aliquoted and stored at −80°C. A tenfold serial dilution 102 of the samples (clinical samples and tissue homogenates) was performed in DMEM supplemented with FCS 103 and penicillin/streptomycin. Inoculum was added in each well and the plate was incubated at 37°C in 5% C02 104 for 1 hour. At least one uninfected well was used as an independent negative control. One hour later, plaque 105 assays were overlaid with carboxy-methylcellulose mix (CMC 3.2% DMEM 5% FBS (V/V)) and after 5 days 106 were fixed and stained with a crystal violet solution (3.7% formaldehyde, 0.2% crystal violet). The titer in 107 PFU/mL (number of plaques / (dilution x volume of diluted virus added to the well) was determined by dividing 108 the number of plaques for the adequate dilution by the total dilution factor. Illkirch, France) using the protocol described by Corman et al. (16) ]. Coronavirus primers (E_Sarbeco_F and 116 E_Sarbeco_R) and probe (E_Sarbeco_P1 labelled with the fluorescent dye FAM-BHQ1) targeting the envelope 117 protein gene (E gene) were used for this study. Primers and probe provided by Eurogentec (Angers, France).

All TaqMan RT-qPCR assays were performed on the thermocycler Rotor Gene Q MDx (Qiagen, Courtaboeuf, 119 France) and Lightcycler LC480 (Roche, France). Negative and positive controls were included in each RT-120 qPCR assay. Positive controls were performed for each assay by testing in duplicates 5 serial 10-fold dilutions 121 of a calibrated SARS-CoV-2 RNA titrating 3.10 6 copies/µL of RNA. The determination of SARS-CoV-2 RNA 122 titer in number of copies/µL was determined by testing a standard curve with six 10-fold dilutions of a SARS-123 CoV-2 RNA titrating 3.10 6 E gene copies/µL of RNA extracted from the SARS-CoV2 strain UCN19 and itself 124 quantified using an E gene transcript. A threshold setting (Ct) of 0.05 was used as the reference for each RT-125 qPCR assay. The efficiency, slope and correlation coefficient (R 2 ) were determined with the Rotor Gene 126 software. All reactions were carried out as technical duplicates. A cut-off > 35 was defined for low positive 127 results (>300 copies/µL of RNA) 128

Samples from the right cranial and caudal lung lobes together with tonsil (in ferrets only) and trachea were 130 fixed by immersion in 10% neutral-buffered formalin and processed routinely into paraffin wax. Four µm 131 sections were cut and stained with haematoxylin and eosin (H&E) and examined microscopically. In addition, 132 samples were stained using the RNAscope in situ hybridization (ISH) technique to identify the SARS-CoV-2 133 virus RNA as previously described (9). Briefly, tissues were pre-treated with hydrogen peroxide for 10 mins 134 (RT), target retrieval for 30 mins (98-101°C) and protease plus for 30 mins (40°C) (Advanced Cell Diagnostics).

A V-nCoV2019-S probe (Advanced Cell Diagnostics, Biotechne) was incubated on the tissues for 2 hours at 136 40°C. Amplification of the signal was carried out following the RNAscope protocol using the RNAscope 2.5 137 HD Detection kit -Red (Advanced Cell Diagnostics, Biotechne). In hamsters, infectious virus was detected from nasal turbinates, trachea and lungs in all the necropsied 198 hamsters at D2 and D4 with 2.7.10 0 to 1.32.10 4 pfu/ml tissue and from oral swabs for two out of three hamsters 199 at D2 (4.10 1 and 2.4.10 2 pfu/ml) and one at D4 (8.0.10 1 pfu/ml). In ferrets, infectious virus was detected in 200 turbinates and nasal washes, but not in the lungs. Nasal turbinates were found infectious (in four out of nine 201 7 of 15 samples) between D2 and D7 with a maximal titer of 1.28.10 4 pfu/ml at D7. Almost all nasal washes (five out 202 six samples) were found infectious between D2 and D4 with a maximal titer of 3.04. 10 3 pfu/ml at D2. 

In experimentally infected hamsters, the clinical signs are generally considered limited, except for the 254 significant weigh loss often reported. In our study, all hamsters tended to gain weight overtime although at 255 different rates, in spite of local evidence of infection. Inoculated ferrets generally present even milder clinical 256 signs of disease, independently from the challenge dose (8-11), without any weight loss, as reported in our 257 study. The marked lethargy observed in ferrets in this study was also reported by others (8-11). It is possible 258 that transient hyperthermia was missed if shorter than 24h (time interval between SC IPTT300 chips reading).

The kinetics, distribution and levels of RNA recovered in hamster tissues were similar to those reported 260 in other studies (6,7,12), including with higher challenge doses, and generally higher than in ferrets, except in 261 the nasal turbinates and large intestine where both species appeared to be on average infected at similar levels. 293 SARS-CoV-2 RNA was also measured in our study in the intestinal tract of ferrets and hamsters, although 294 at a lower level than in the upper respiratory tract. As in most human cases (22), no infectious virus was isolated 295 from intestinal samples, while viral RNA was detected. The presence of SARS-COV-2 in the brain has also 296 been reported in other experimental studies (6,10,11), and rarely in human patients, although not systematically.

In our study, viral RNA was recovered in both species mostly in the olfactive bulb but also in the spinal bulb.

The impact of this dissemination in unclear.

Antibody kinetics in ferrets and hamsters were comparable with 1/ those seen in patients with a 300 seroconversion between D7 and D14 (22), and 2/ those seen in experimental models using hamsters and ferrets 301 (6, (8) (9) (10) (11) . In our study, the increase in antibody levels occurred faster in hamsters than in ferrets, from no 302 detectable level at D4 to consistently high levels between D7 and D14 in hamsters and between D7 and D10 in 303 ferrets. These observations suggest that in these species, the exposure to low challenge viral dose inoculated by 304 the intranasal route induced an early, significant and potentially protective humoral response. For both species, 305 the rise of antibody levels slightly preceded or was concomitant with the disappearance of infectious virus (viral 306 clearance) but further investigations would be required to conclude on the protective effect conferred by 307 antibodies.

According to previous studies (9), reducing the challenge dose beyond the one used in our study impacts 309 the reproducibility of infection success (only one out of six ferrets inoculated with 5.10 2 pfu became infected 310 in Ryan et al., experiments (9) ). Infection doses should also be expressed and standardized per animal weight.

In our study, we followed standard protocols by delivering homogeneous challenge doses between animals. As 312 a result, the dose per weight was in the order of ten times higher in hamsters than in ferrets, which can contribute 313 to the higher pathogenicity observed in this species. In addition, the females received a larger dose/kg than 314 males since the same bolus was delivered to all animals in spite of different weights (average weight of 1419 g 315 for males and of 740 g for females at D0). A lower susceptibility has been suggested in females, at least in 316 humans (23-26), and this may explain why no difference in the severity of the disease was measurable between 317 sexes in our study. Weight dependent infection doses in future studies may highlight sex differences in the 318 severity of experimental outcomes.

COVID-19 is less frequently reported in children than in adults and elderly people (27)]. Two studies in 320 hamsters (12,13) reported more severe lesions and clinical signs in older animals. The oldest ferrets used in 321 experimental studies were 20 month old (8-11)], therefore no conclusion can be drawn on the effect of age in 322 10 of 15 this species. Some studies suggest that thymus produced thymosin may have protective effect (28, 29) ]. The 323 youngest animals included in our study still presented a detectable active thymus at post-mortem but its activity 324 and influence on the disease pattern was not explored.

Hamsters and ferrets are valuable animal models to study COVID-19. Hamsters are easy to handle and maintain.

Ferrets are more expensive than hamsters and require larger housing space, but a change in their generally active 328 behaviour is a relatively easy clinical sign to detect in comparison with hamsters. The marked transient fatigue 329 reported in the infected ferrets at days 7 and 8 was also reported by others (8, 9, 11) 

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Figure 1. Hamsters weight changes from D0 (%) with mean and standard deviation after inoculation with 438 SARS-CoV-2 or PBS

Figure 2. Viral RNA loads in ferrets and hamsters inoculated with UCN19 SARS-CoV-2

Figure 3. Viral RNA loads in clinical samples. (A): oral swabs, (B): nasal washes, (C): rectal swabs 444 collected from to D2 to D14 in three ferrets and three hamsters inoculated intra-nasally with UCN19 445 SARS-CoV-2

Inflammatory infiltrates with the lung parenchyma, 451 mostly within the bronchial and bronchiolar mucosa but also surrounding airways and blood vessels are 452 observed at D2 (C, 100x) and D4 (E, 200x). The presence of the inflammatory infiltrates is correlated with 453 the viral RNA staining