# Infectious potential and circulation of SARS-CoV-2 in wild rats

Kevyn Beissat, Virginie Lattard, Evelyne Picard-Meyer, Ambre Fafournoux, Sionfoungo Daouda Soro, Alexandre Servat, Françoise Vincent-Hubert, Franck Boué, Nolan Chatron, Elodie Monchâtre-Leroy, et al.

► To cite this version:

Kevyn Beissat, Virginie Lattard, Evelyne Picard-Meyer, Ambre Fafournoux, Sionfoungo Daouda Soro, et al. Infectious potential and circulation of SARS-CoV-2 in wild rats. 2024. anses-05067585

HAL Id: anses-05067585

https://anses.hal.science/anses-05067585v1

Preprint submitted on 14 May 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 Infectious potential and circulation of SARS-CoV-2

2 in wild rats

3 Wild rats and SARS-CoV-2

4 Kevyn Beissat¹,²*, Virginie Lattard², Evelyne Picard-Meyer¹, Ambre Fafournoux²,

5 Sionfoungo Daouda Soro², Alexandre Servat¹, Françoise Vincent-Hubert³, Franck Boué¹,

6 Nolan Chatron², Elodie Monchâtre-Leroy¹, Marine Wasniewski¹*

7 ¹ Nancy Laboratory for Rabies and Wildlife, ANSES, Malzéville, France

8 ² USC-1233 Rongeurs Sauvages Risques Sanitaires et Gestion des Populations (RS2GP),

9 VetAgroSup, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Lyon University, Marcy-L'Etoile, France

11 ³ IFREMER, Laboratoire Santé, Environnement et Microbiologie (LSEM) - MASAE, rue de l'île d'Yeu, BP 21105, 44311 Nantes, France / Groupement d'intérêt scientifique (GIS)

13 Obépine, https://www.reseau-obepine.fr

14 * Corresponding authors

15 E-mail: kevyn.beissat.ext@anses.fr (KB), marine.wasniewski@anses.fr (MW)

16 Abstract

17 Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-

18 2) pandemic, a wide range of animal species (pets, mink...) have been naturally infected

19 with this betacoronavirus. The emergence of new variants has increased the ability of
20

SARS-CoV-2 to infect species that were not susceptible to the “original” SARS-CoV-2, such as mice and rats.

21

22

This work attempted to evaluate the role of urban rats in the SARS-CoV-2 transmission by combining surveillance studies of rat populations in urban environments, in vivo experimental inoculation of SARS-CoV-2 and comparative viral-receptor interaction in silico analyses.

23

24

25

26

We studied the circulation of SARS-CoV-2 in wild *Rattus norvegicus* (n=401) captured in urban areas and sewage systems of several French cities. Except for 3 inconclusive samples (2/75 from Bordeaux and 1/261 from Lyon) none of the 353 sera tested showed anti-SARS-CoV-2 antibodies by microsphere immunoassay. However, the 3 inconclusive sera samples were negative by virus neutralisation assay. No SARS-CoV-2 viral RNA was detected in all lungs collected from the 401 captured urban brown rats. In complement, four rat groups (two wild-type colonies, *Rattus norvegicus* and *Rattus rattus*, and two laboratory strains, Sprague-Dawley and Wistar) were inoculated with the SARS-CoV-2 Omicron BA.5. At 4 days post-inoculation, no infectious viral particles were detected in the lungs and upper respiratory tract (URT) while viral RNA was detected at a low level only in the URT of all groups. In addition, seroconversion was observed 14 days after inoculation in the four groups. By molecular modelling, the Omicron BA.5 receptor binding domain (RBD) had lower affinities for *Rattus norvegicus* and *Rattus rattus* ACE2 than *Homo sapiens* ACE2.

27

28

29

30

31

32

33

34

35

36

37

38

39
40 Based on these results the SARS-CoV-2 Omicron BA.5 was unable to infect laboratory and
41 wild type rats. In addition, *Rattus norvegicus* collected for this study in different areas of
42 France were not infected with SARS-CoV-2.

43 **Keywords:** SARS-CoV-2, Omicron BA.5, urban rodents, wild rat, *Rattus norvegicus*, *Rattus rattus*

45 **Introduction**

46 In late December 2019, a novel coronavirus called severe acute respiratory coronavirus 2
47 (SARS-CoV-2) was identified as the causative agent of COVID-19 disease in Wuhan, China
48 [1]. This virus, which belongs to the betacoronavirus genus, rapidly spread in the human
49 population and caused numerous deaths with serious repercussions on global health.
50 Shortly after the onset of the pandemic, the first cases of natural animal infections
51 occurred [2]. Indeed, several animal species, including pets (dogs and cats), farm animals
52 (minks), zoo animals (tigers and lions), and wild animals (otters and white-tailed deer)
53 were reported to be naturally infected with this coronavirus [3–8]. This phenomenon
54 warrants careful consideration as it may increase the risk of viral mutation and
55 subsequent transmission of potentially more pathogenic viruses to humans. This
56 phenomenon has already been observed in mink and white-tailed deer species [9,10].

57 Mice and rats were not susceptible to infection with the "original" SARS-CoV-2 [11-13].
58 However, the emergence of new variants of concern (VOCs) such as Alpha (B.1.1.7), Beta
59 (B.1.351) and Omicron (B.1.1.529, BA.5...) variants with favourable mutations in the spike
60 protein seems to modify the viral host range of this virus. Indeed, infectious viral particles
61 have been detected in the nasal turbinates and lungs of C57BL/6J mice and Sprague-
62 Dawley rats infected with the Alpha, Beta or Delta (B.1.617.2) VOC [12,14,15]. In addition,
63 it was demonstrated that the Delta variant is unable to infect wild mice, whereas the Beta
64 variant can spread through close contact between infected and naive BALB/c mice [16]
65 and between Sprague-Dawley rats [14]. These results suggest that mutations in the spike
66 protein such as the N501Y mutation may enhance the ability of the virus to infect new
67 species, including rodents.

68 The SARS-CoV-2 genome has been widely found in wastewater [17], an environment,
69 where wild rats roam freely. These findings raise concerns about the potential
70 transmission of the virus to urban rodents. While the presence of infectious SARS-CoV-2
71 particles in wastewater has not yet been demonstrated, a recent experimental study,
72 highlighted the survival of SARS-CoV-2 infectious particles in unfiltered/filtered raw
73 sewage, and secondary effluent for several hours (between 10 and 18 hours) [18]. The
74 emergence of new variants and the circulation of SARS-CoV-2 viral RNA in sewage level
75 up the risk of infection of urban rats. Therefore, the possibility of urban rodents becoming
76 a new animal reservoir for the virus, with potential spillover to humans, should not be
77 overlooked. In addition, a study conducted on wild rats captured in urban areas of the
78 USA, close to wastewater, detected partial SARS-CoV-2 genome in the lungs of *Rattus
79 norvegicus* [15]. The presence of antibodies against SARS-CoV-2 has also been
demonstrated in brown rats (*Rattus norvegicus*) in the USA, the United-Kingdom and
80 Canada [15,19,20].
81
82 In this context, this work attempted to evaluate the role of wild rats in the SARS-CoV-2

83 transmission by combining surveillance studies of rat populations in urban environments

84 across various French cities, in vivo experimental inoculation of SARS-CoV-2 into both wild

85 and laboratory rats and comparative viral-receptor interaction in silico analyses.

86 **Results**

87 **Field monitoring of SARS-CoV-2 infections in urban rat populations**

89 A total of 401 wild rats were collected from six French cities (Fig 1 and Table 1), distributed

90 homogenously across the country, with human populations ranging between 200,000 to

91 1,500,000 inhabitants and average population densities between 2,000 and 10,000

92 inhabitants per square kilometre: Besançon (4.7% of the rats), Bordeaux (19.2%), Lyon

93 (67.1%), Marseille (3.0%), Nancy (5.0%) and Nantes (1.0%) between January 2022 and July

94 2023 (Fig 2). These rats were trapped in sewers (29.2%), in social housing courtyards with

95 direct connection to the sewer network (58.3%) or in urban parks without connection to

96 the sewer network (12.5%). Within the sample, 57.2% were males and the median weight

97 of trapped rats was 266.7g (95% CI: 243.8-287.9g), with values ranging from 26g to 600g

98 (excluding Marseille rats, which could not be weighed) (Table 1). All the 401 wild rats have

99 been confirmed to belong to the species *Rattus norvegicus* through morphometric

100 measurements, as well as by amplification and sequencing of the cytochrome b gene in

101 cases of uncertainty.

102
<table><caption>Table 1. Characterization of wild urban rat populations captured in these cities as part of the surveillance study on SARS-CoV-2 infection in rats.</caption><thead><tr><th rowspan="3">Urban area</th><th rowspan="3">Number of rats</th><th colspan="4">Type of site</th><th rowspan="3">Weight</th><th rowspan="3">% males</th></tr><tr><th rowspan="2">Sewer</th><th colspan="2">Surface</th></tr><tr><th>Connected to sewers</th><th>Not connected to sewers</th></tr></thead><tbody><tr><td>Besançon</td><td>19</td><td>19</td><td>-</td><td>-</td><td></td><td>207 [161-230]</td><td>58</td></tr><tr><td>Bordeaux</td><td>77</td><td>-</td><td>77</td><td>-</td><td></td><td>200 [147-269]</td><td>55</td></tr><tr><td>Lyon</td><td>269</td><td>74</td><td>148</td><td>47</td><td></td><td>298 [272-314]</td><td>60</td></tr><tr><td>Marseille</td><td>12</td><td>-</td><td>9</td><td>3</td><td></td><td>ND</td><td>25</td></tr><tr><td>Nancy</td><td>20</td><td>20</td><td>-</td><td>-</td><td></td><td>77 [41-148]</td><td>55</td></tr><tr><td>Nantes</td><td>4</td><td>4</td><td>-</td><td>-</td><td></td><td>294 [175-360]</td><td>75</td></tr><tr><td>N</td><td>401</td><td>117</td><td>234</td><td>50</td><td></td><td></td><td></td></tr><tr><td>Weight</td><td>267 [244-288]</td><td>236 [218-256]</td><td>303 [269-340]</td><td>258 [201-292]</td><td></td><td></td><td></td></tr><tr><td>% males</td><td>57</td><td>60</td><td>53</td><td>70</td><td></td><td></td><td></td></tr></tbody></table>
106 401 lungs and 353 sera (75 from Bordeaux, 261 from Lyon, 12 from Marseille and 5 from Nancy) were collected from the 401 rats captured. All animals were found to be RT-PCR negative for SARS-CoV-2 in the lungs, while wastewaters collected in sewers were positive depending on the city (Besançon Ct = 28 – 37; Lyon = 33- 35; Nantes= 28 – 36; Nancy = 34 – 40). Presence of anti-SARS-CoV-2 antibodies were thus screened in 353 sera (for some rats, blood was not obtained) by MIA. To determine the cut-off of positivity, the entire rat population was used, considering the low percentage of natural infection of *Rattus norvegicus* and the absence of pre-pandemic sera [21]. None of the 353 tested sera were positive for the three SARS-CoV-2 antigens and inconclusive results were obtained for 3 samples (Fig 2). Two of these samples (R701 and R775) were collected in Bordeaux, from rats trapped in a social housing courtyard with direct connection to the sewer network, and the third sample (R697) was collected in Lyon, on a rat trapped in a sewer. These 3 samples were collected in June 2023 when the XBB 1.5 variant circulated in French human population [22]. Samples having inconclusive result were tested by seroneutralisation (SN) assay using the SARS-CoV-2 D614G as the challenge virus. All samples were found negative.

122

123 **Fig 2. Detection of sera anti-SARS-CoV-2 antibodies in the urban rats using microsphere immunoassay**
124 (MIA).

125 Antibodies were detected using three SARS-CoV-2 antigens (the nucleoprotein, the subunit 1 (S1) receptor
126 binding domain (RBD) of the spike and the trimeric spike). Antibody levels were expressed as Relative
127 Fluorescence Intensities (RFI) to control antigen. The negative group (the whole population) was used to
128 determine the cut-off (mean + 3 standard deviation). In all graphs, mean-values are presented.
129 Seroconversion was defined as the detection of antibodies against the three antigens, while results were
130 considered inconclusive if antibodies were detected against two of the three antigens.

131 **Experimental in vivo inoculation of SARS-CoV-2 in laboratory and**
132 **wild rats**

133 Concurrently, we performed *in vivo* experiments to evaluate the susceptibility of both
134 laboratory and wild rats to Omicron BA.5 variant inoculation. Since no significant
135 differences in response were observed between the three inoculated wild *Rattus*
136 *norvegicus* colonies (A, B and C), data from these colonies were combined for simplified
137 graphical representation.

138 **Clinical signs**

139 No clinical signs or significant variations in weight (p>0.05) were observed between the
140 negative groups and the Omicron BA.5-inoculated groups at 4 and 14 dpi whether in
141 laboratory rats or wild *Rattus norvegicus* or *Rattus rattus* (Fig 3).

142

143 **Fig 3. Body weight evolution of the PBS inoculated rats versus the Omicron BA.5 inoculated rats for the**
144 **four groups.**

145 The means are represented ± one standard deviation. Data for Sprague-Dawley at D14, are missing. PBS:
146 groups control.
147 Detection of viral RNA, infectious viral particles and antibodies anti-SARS-CoV-2 in inoculated rats

149 Viral RNA was detected at 4 dpi in the upper respiratory tract (URT) of all groups, including both wild and laboratory rats (Fig 4). The mean viral load for Sprague-Dawley and Wistar rats was approximately 3.3x10² and 4.1x10² RNA copies/µL of URT supernatant, respectively. For wild groups, it was 8x10² and 8.8x10³ RNA copies/µL of URT supernatant from *Rattus norvegicus* and *Rattus rattus*, respectively. No significant differences (p>0.05) were observed between any of the groups. In addition, SARS-CoV-2 RNA was not detected in the lungs of any of the rats. Furthermore, no infectious viral particles were detected in cell culture by TCID50 assays at 4 dpi in the lungs or the URT of any of the four groups of rats.

158

159 Fig 4. Detection of viral RNA load in the upper respiratory tract (URT) of the four rat groups at 4 days post infection (D4).

160

161 The means are represented ± one standard deviation.

162 The presence of anti-SARS-CoV-2 antibodies in plasma was investigated by MIA at 4 and 14 dpi. At 4 dpi, no seroconversion was observed in any group of rats, whereas at 14 dpi, antibody responses were observed in the four groups of rats. However, while all Sprague-Dawley and Wistar rats seroconverted, seroconversion was only detected in 4/8 wild *Rattus norvegicus* and 4/7 *Rattus rattus* (Fig 5). To investigate the neutralisation efficiency of the detected anti-SARS-CoV-2 antibodies, a seroneutralisation (SN) assay using the Omicron BA.5 as challenge virus was performed on all plasmas (Fig 6). Four wild *Rattus*

163

165

166

167

168
169 *norvegicus*, one *Rattus rattus* and five Sprague-Dawley and Wistar showed neutralising 170 anti-SARS-CoV-2 antibodies up to 1/90 dilution. For both the MIA and SN methods, no 171 antibodies were detected in the negative group. In the two laboratory rat strains and the 172 *Rattus rattus* colony, the results showed that rats positive by SN, were also detected 173 positive by MIA. In the wild *Rattus norvegicus* colony, seroconversion was observed in 174 two rats (R5 and R11) by SN whereas no antibodies were detected by MIA. (Table 3). 175 Specifically, these two samples, defined as inconclusive results, were tested positive for 176 two out of the three SARS-CoV-2 antigens (nucleoprotein and spike trimeric antigens). In 177 addition, all wild *Rattus norvegicus* and *Rattus rattus* sera were positive or inconclusive 178 results. 179

180 **Fig 5. Detection of anti-SARS-CoV-2 antibodies in plasma at 4 and 14 dpi by microsphere immunoassay in**
181 **the four groups of rats.**

182 Three SARS-CoV-2 antigens (the nucleoprotein, the subunit 1 (S1) receptor binding domain (RBD) of the 183 spike and the trimeric spike) were used. The levels of anti-SARS-CoV-2 antibodies were expressed as 184 Relative Fluorescence Intensities (RFI) to the control antigen. A: *Rattus norvegicus* (wild), B: *Rattus rattus*, 185 C: Sprague-Dawley and D: Wistar. The negative group was used to determine the cut-off (mean + 3 standard 186 deviation). For all graphs, the means are represented ± one standard deviation. The blue background 187 represents the seropositivity area. Seroconversion was established when antibodies were detected with 188 the three antigens and plasma was considered inconclusive when two of the three antigens were positive. 189

190 **Fig 6. Levels of neutralising antibodies detected in the four groups of rats.**
191 *Rattus norvegicus* (wild) (blue) (n=8), *Rattus rattus* (green) (n=8), Sprague-Dawley (orange) (n=7) and Wistar (purple) (n=7) at 14 dpi. Animals from negative group are indicated by a black circle. Each point represents one rat and the mean-values are represented on the graph.

194

195 **Table 2.** Comparison of the results obtained with the microsphere immunoassay and the seroneutralisation methods for the four groups of rats.

196

<table><thead><tr><th rowspan="2">Group</th><th rowspan="2">Method</th><th colspan="10">Rat identifier</th></tr><tr><th>R4</th><th>R5</th><th>R6</th><th>R7</th><th>R8</th><th>R9</th><th>R10</th><th>R11</th></tr></thead><tbody><tr><td rowspan="2">Rattus norvegicus (wild)</td><td>MIA</td><td>Yes</td><td>Inc</td><td>Yes</td><td>Yes</td><td>Inc</td><td>Inc</td><td>Yes</td><td>Inc</td></tr><tr><td>SN</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Yes</td></tr><tr><td colspan="2"></td><td>R11</td><td>R12</td><td>R13</td><td>R14</td><td>R15</td><td>R16</td><td>R20</td><td>-</td></tr><tr><td rowspan="2">Rattus rattus</td><td>MIA</td><td>Inc</td><td>Inc</td><td>Yes</td><td>Inc</td><td>Inc</td><td>Yes</td><td>Yes</td><td>-</td></tr><tr><td>SN</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>-</td></tr><tr><td colspan="2"></td><td>R14</td><td>R15</td><td>R16</td><td>R17</td><td>R18</td><td>R19</td><td>R20</td><td>-</td></tr><tr><td rowspan="2">Sprague-Dawley</td><td>MIA</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>-</td></tr><tr><td>SN</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>Yes</td><td>Yes</td><td>-</td></tr><tr><td colspan="2"></td><td>R14</td><td>R15</td><td>R16</td><td>R17</td><td>R18</td><td>R19</td><td>R20</td><td>-</td></tr><tr><td rowspan="2">Wistar</td><td>MIA</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>-</td></tr><tr><td>SN</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td><td>-</td></tr></tbody></table>

197 MIA: Microsphere immunoassay and SN: seroneutralisation. "Yes": presence of a seroconversion, "No":

198

absence of a seroconversion and "Inc": inconclusive results (for MIA results presented in Fig 5).
199 Modelling of the interaction between the RBD of Omicron BA.5
200 and the ACE2

201 The percentage of identity (amino acids sequences) between angiotensin-converting
202 enzyme 2 (ACE2) of both *Rattus* species was 96%. The *Homo sapiens* ACE2 shared
203 respectively 82% and 79% of identity with *Rattus norvegicus* and *Rattus rattus* (S1 File).
204 Positions of the Omicron BA.5 receptor binding domain (RBD) relative to ACE2 receptors
205 of *Homo sapiens*, *Rattus norvegicus* and *Rattus rattus* are showed on Figs 7A-C. The
206 affinity of each complex was estimated using PMF calculations based on molecular
207 dynamics simulations, which required separating Omicron BA.5 from ACE2. Complete
208 separation of both proteins were visualised by a plateau (Fig 7D), the resulting binding
209 energy values for *Homo sapiens*, *Rattus norvegicus* and *Rattus rattus* were respectively
210 45 ± 1, 28 ± 1 and 33 ± 1 kcal/mol.

211

212 Fig 7. Modelling of the interaction between the receptor binding domain (RBD) of SARS-CoV-2 Omicron BA.5 and the ACE2 of *Homo sapiens*, *Rattus norvegicus* and *Rattus rattus*.

213 3-dimensions structures showing receptor cellular ACE2 of *Homo sapiens* (blue) (A), *Rattus norvegicus* (orange) (B) and *Rattus rattus* (pink) (C) interacting with the receptor binding domain (RBD) of Omicron BA.5 (green). Cell membrane is representing in grey. (D) ACE2/RBD affinities in kcal/mol during separation of both proteins with *Homo sapiens* in blue, *Rattus norvegicus* in orange and *Rattus rattus* in pink.
218 **Discussion**

219 Our study primarily investigates the circulation of SARS-CoV-2 in wild rats, *Rattus norvegicus*, in France. Trapping was conducted in multiple cities throughout France,
220 including Besançon, Nancy, Bordeaux, Lyon, Marseille, and Nantes, with the objective of
221 enhancing the representativeness of the study population. Of the 401 rats included in the
222 study, 351 were captured in areas connected to the sewer system, with 117 of these being
223 captured directly within sewers. Sampling efforts were concentrated in sewers or areas
224 in close proximity to sewers due to the extensive report of the SARS-CoV-2 genome in
225 wastewater [23], which may serve as a potential source of infection for rats in close
226 contact with contaminated water. The sampling was conducted between January 2022
227 and July 2023, a period during which several SARS-CoV-2 variants were circulating in
228 human population (i.e. Omicron BA.2, BA.5, XBB 1.5 and EG.5). In addition, the effective
229 presence of SARS-CoV-2 in the wastewater from the various trapping areas was confirmed
230 to be present. The circulation of SARS-CoV-2 in rats was assessed using a dual approach
231 that combined molecular biology techniques for detecting specific viral RNA in lungs, with
232 serological analysis to detect specific anti-SARS-CoV-2 antibodies.
233

234 No traces of SARS-CoV-2 viral RNA were detected in any of the 401 lungs samples by RT-
235 PCR, indicating the absence of viral replication in the lungs of the trapped rats. This finding
236 is significant, as the lungs are the tissue where viral replication would typically be
237 expected, and thus these results rule out the presence of an ongoing SARS-CoV-2
238 infection in these rats, despite the presence of the virus genome in the wastewater.
Similar results have been observed in previous studies, which also reported the absence of viral RNA in the lungs of wild rats captured in Belgium, Germany and Spain [24–26], as well as in other matrices, including feces, oronasopharyngeal swabs, nasal turbinates, intestines, and various other tissues [20,27,28]. Despite these results, infection of rats by SARS-CoV-2 seems possible, as a few rare studies have reported positive cases. For instance, four out of 79 rats tested positive in the United States, and three out of 72 in Mexico [15,29], but it should be noted that these results were obtained using RT-PCR methods different from the one carried out in our study (different targeted genes and primers). Several hypotheses can be put forward to explain our findings. Either SARS-CoV-2 was undetectable within rat populations in contact with wastewater during the study period. Or the virus present in the wastewater was not viable or infectious [30], which would prevent the rats from becoming contaminated, despite a recent study indicating that the virus can survive for a few hours in wastewater under experimental conditions [18]. Another possibility is that, although the rats were exposed to the virus, it was unable to replicate efficiently in their tissues due to mutations in the spike protein that could affect its affinity for the cell entry receptor or alter its replication in rodent, as previously shown for the SARS-CoV-2 B.1.351 variant [14], the B.1.1.529 Omicron (BA.1) [31,32] or the BA.5.5 Omicron variants [15].

To explore this further, serological analyses were conducted in parallel to detect potential signs of past infection. Of the 353 serum samples from *Rattus norvegicus*, three produced inconclusive results, testing positive for two out of three SARS-CoV-2 antigens when
261 analyzed using a multiplex microsphere immunoassay. The three inconclusive samples 262 later tested negative in the neutralization assay, confirming the absence of neutralizing 263 SARS-CoV-2 antibodies in these rats. This may be explained by the absence of exposure 264 of the trapped wild rats to infectious virus in wastewater, or by low seroconversion and 265 the lack of maintenance of antibodies over time. Another possibility should be that the 266 antibodies produced by the SARS-CoV-2 variants circulating in wastewater at the time of 267 the study are not able to neutralise the variant D614G used as challenge virus in our 268 neutralisation assays. Indeed, as recent findings demonstrated that Omicron variants 269 escape serum neutralisation to a greater extent than the ancestral D614G strain, we can 270 put forward the opposite hypothesis that the D614G variant could be insensitive to 271 antibodies raised against the new Omicron variants.

272 Our findings are in concordance with those of Colombo *et al*. obtained at the beginning 273 of the pandemic, who reported that 3 out of 35 *Rattus norvegicus* were positive by MIA 274 but were all negative by viral neutralisation test [24]. In addition, no SARS-CoV-2 275 antibodies were detected in studies conducted on wild rat populations in Germany, 276 Belgium/France and Spain [25,26,33]. Conversely, the presence of antibodies by 277 seroneutralisation assays has been reported in *Rattus sp.* in the USA (2 out of 117 *Rattus 278 norvegicus*), Hong-Kong (1 out of 217 *Rattus norvegicus*), Canada (2 out of 213 *Rattus 279 norvegicus*), Germany (1 out of 130 *Rattus norvegicus*) and Malaysia (1 out 3 *Rattus 280 rattus*) [20,27,28,34,35]. However, some studies detected antibodies using other 281 methods for detecting binding antibodies such as ELISA but reported negative results by 282 neutralisation assays [15,19]. Seroneutralisation results can vary depending on the
283 method used (with live virus or purified proteins) [36]. Indeed, studies demonstrating 284 positive neutralising antibody samples surrogate virus neutralising test (sVNT) using 285 proteins (mainly RBD) instead of live virus such as in our study. Furthermore, sera positive 286 by MIA or ELISA were negative by conventional surrogate virus neutralising test (cVNT) 287 using live virus [15,24]. cVNT is the gold standard to detect neutralising antibodies and a 288 high correlation between these methods has been demonstrated [37].

289 Regardless of the method used, cross-reactivities of the detected SARS-CoV-2 antibodies 290 with other coronaviruses circulating in rats cannot be excluded, as recently demonstrated 291 for human and other animals [38]. This phenomenon was evoked for SARS-CoV-2 292 [15,20,28] and for coronaviruses detected in wild rodents [39].

293 In order to advance our understanding of the epidemiological role of wild rats – whether 294 they are unexposed, unresponsive and or insensitive – we inoculated wild rats (Rattus 295 norvegicus and Rattus rattus) and laboratory rats (Sprague-Dawley and Wistar) with the 296 SARS-CoV-2 Omicron BA.5. This strain was the main SARS-CoV-2 variant that circulate 297 during the sampling of wild rats.

298 No clinical signs or body weight loss were observed in the inoculated groups in 299 comparison to the controls. These findings align with those of previous in vivo studies 300 conducted on Sprague-Dawley rats infected with various SARS-CoV-2 variants, including 301 the Wuhan-like strain, Alpha, Beta, Delta, and Omicron (BA.5) [12,14,15]. In our 302 experiments, only viral RNA was isolated in the URT at 4 dpi, with RNA titers ranging 303 between 4.7x10³ and 3.2x10⁵ RNA copies/mg of tissue, while no viral RNA was detected 304 in the lungs, and no infectious particles (TCID50 assays) were detected in any tissue (lungs
or URT). Our results in URT samples could be due to the difference of sensitivity between RT-qPCR and TCID50 assay (cell culture). Indeed, in our laboratory, the thresholds of detection are 10² TCID50/mL and 25 copies/µL for the TCID50 assays and RT-qPCR tests respectively. It is also important to note that in our study, all animals were inoculated with a high dose of virus (titers ranging from 10⁵.⁹⁷ to 10⁶.⁷⁴ TCID50/mL (approximately 4.9x10⁶ to 5.5x10⁶ RNA copies/µL)), and viral RNA titers found in the URTs were systematically lower (between 3.3x10² and 8.8x10³ RNA copies/µL of URT supernatant) than these virus doses. It could therefore also be assumed that only remnants of viral RNA or viral particles from the inoculum were detected by RT-qPCR at 4 dpi which could help to explain the absence of infectious virus in URT and lungs and viral RNA in lungs. These results provide further evidence to support the hypothesis that either viral replication occurred at a very low level in these tissues, or was abortive or absent. Discrepant results were observed in the existing literature by using a different Omicron variant. Indeed, for Omicron BA.5.5 variant, Wang *et al*. detected infectious virus only at 2 dpi in the lungs of Sprague-Dawley rats, inoculated with 2.10⁴ PFU/rats of virus, while no infectious particles were detected in URT [15]. In addition, in their study, viral RNA was detected at both 2 and 4 dpi in lungs and URT at a level demonstrating efficient replication [15].

Regarding the humoral immune response to the infection with Omicron BA.5 variant by intranasal (IN) route, it shall be noted that antibodies were detected in all animals of the two laboratory strains, whereas seroconversion was only observed in four of the eight wild *Rattus norvegicus* and four of the seven *Rattus rattus*. These differences between
327 wild rats and laboratory rats can be attributed to the immune system specific to each of

328 these models. In the case of wild rats, we need to talk about eco-immunology, because

329 their immune response is conditioned by the environment and the external stimuli they

330 are confronted with on a daily basis. As a reminder, the three key concepts of eco-

331 immunology are immuno-heterogeneity, sub-maximal immune response due to energy

332 limitation and the diversity of the antigenic load with which animals may be confronted.

333 In the case of laboratory animals, they are bred under controlled conditions.

334 Immunoheterogenicity is therefore limited, energy resources are not limited to maximise

335 immune response and contact with external pathogens is kept to a minimum. Taken

336 together, these factors could optimise their immune response, unlike wild animals [40].

337 The results obtained in the present study are in agreement with those reported by Wang

338 et al., demonstrating the presence of IgG and neutralising antibodies at 21 dpi [15].

339 These results obtained for the in vivo manipulations, even for wild rats, are inconsistent

340 with those obtained in the field. Indeed, contrary to the results obtained in our field study,

341 the presence of anti-SARS-CoV-2 antibodies was confirmed in all four groups of rats at 14

342 dpi, using both MIA and serum neutralisation tests despite the absence of virus replication

343 in the target tissues. A phenomenon of more or less marked passive immunisation of

344 animals in response to the presence SARS-CoV-2 viral particles inoculated by IN route

345 could explain the antibody production. This finding could also suggest that the trapped

346 rats during our field study were not exposed to infectious particles of SARS-CoV-2 in

347 sewers as no antibodies were detected.

348
349 To summarise the results of our *in vivo* studies, no signs of viral infection were detected
350 in wild-type or laboratory rats following inoculation of the Omicron BA.5 variant by the
351 IN route and the antibody response should be considered more as a marker of passive
352 immunisation response than of viral infection.

353 The results obtained from our field and *in vivo* studies suggest that rats have little or no
354 receptivity or susceptibility to infection with SARS-CoV-2 (particularly with the Omicron
355 BA.5 variant). As indicated earlier in the discussion, the low or inefficient replication of
356 the virus demonstrated in this article could be explained by a low affinity between the rat
357 ACE2 receptor (cell entry receptor) and the Omicron BA.5 variant. Indeed, it is well known
358 that virus-receptor interaction is generally an effective determinant of virulence. To
359 explore this hypothesis, molecular modelling has been conducted between the ACE2 of
360 *Rattus norvegicus*, *Rattus rattus* and *Homo sapiens* and the Omicron BA.5 RBD. The
361 findings align with the experimental outcomes, indicating a diminished affinity of the RBD
362 for the ACE2 of both *Rattus* species (28 ± 1 and 33 ± 1 kcal/mol) in comparison to human
363 ACE2 (45 ± 1 kcal/mol). These results offer a partial explanation for the lack of
364 susceptibility of rats to Omicron BA.5. Indeed, a low affinity between the RBD receptor
365 and ACE2 results in a weaker attachment of the virus to the cell. Nevertheless, a
366 discrepancy exists between the results of *in silico* calculations and *in vivo* experiments,
367 particularly with regard to the role of the animal immune system. In the context of the
368 3Rs, molecular modelling could be employed as a replacement for animal
369 experimentation in the first instance. In this context, the evaluation of diverse RBDs from
370 emerging SARS-CoV-2 variants in comparison with the ACE2 of *Rattus* sp. could serve as a
371 pivotal approach to ascertain the potential for these variants to infect rats, obviating the
372 necessity for animal experimentation.

373

374 The findings of this study indicate that wild or laboratory rats do not play an epidemiologic
375 role in the SARS-CoV-2 pandemic, particularly in relation to the Omicron BA.5 variant.

376 Indeed, no evidence of the presence of SARS-CoV-2 RNA was identified in the lungs and
377 sera of rats captured in urban and sewage environments in several French cities.

378 Furthermore, the results of the *in vivo* experiments and *in silico* calculations are in
379 accordance with those of the field study. Indeed, the four groups of rats (wild *Rattus
380 norvegicus*, wild *Rattus rattus*, and laboratory *Rattus norvegicus* Sprague-Dawley and

381 Wistar) exhibited no susceptibility to infection by the SARS-CoV-2 Omicron BA.5. In light
382 of the rapid evolution of the virus, its adaptation to new hosts and the emergence of
383 SARS-CoV-2 variants, it is imperative to continue studying the circulation of SARS-CoV-2
384 in wild rodents in order to investigate the potential establishment of a wildlife reservoir.

385 Furthermore, as a preliminary approach, molecular modelling could be conducted using
386 emerging SARS-CoV-2 variants before proceeding with animal experimentation.

387 **Materials and Methods**

388 **Ethics statements**

389 The experimental protocols involving rats complied with the regulation 2010/63/CE of the
390 European Parliament and of the council of 22 September 2010 on the protection of
391 animals used for scientific purposes [41] and as transposed into French law [42].
392 *In vivo* experimental studies were approved by the Anses/ENVA/UPEC ethics committee
393 and authorised by the French Ministry of Research (Apafis authorization n° #37782-
394 2022062312057722 v4).

395 Field experimental studies were approved by the ethics committee of the Veterinary
396 School of Lyon and authorised by the French Ministry of Research (Apafis authorization
397 n° #37713-2022042712262465).

398 Rat captures and samples

399 Wild rats were captured in sewers and parks connected or not to sewers in several French
400 cities including Besançon, Bordeaux, Lyon, Marseille, Nancy and Nantes between January
401 2022 and July 2023. Chronologically, Omicron BA.2, BA.5, XBB 1.5 and EG.5 circulated in
402 the French population during the sampling period [22]. *Rattus norvegicus* were captured
403 using live traps or obtained through pest control campaigns. The rats were then
404 transported in cages to a laboratory, where they were anaesthetised with isoflurane and
405 euthanised by cardiac puncture and cervical dislocation. Various measurements including
406 sex, weight, height, body length, and tail length were meticulously reported for each
407 animal. Cytochrome b was performed to confirm the rat species in case of uncertainty.

408 Each animal was aseptically autopsied and various tissues, including lung were removed,
409 transferred to sterile tubes, and stored at -80°C until RNA extraction. For viral RNA
410 extraction, lung tissue samples (~20 to 60 mg) were placed in tubes containing beads
411 (Lysing Matrix E, MP Biomedicals™) and 1 mL of Dulbecco's Modified Eagle medium
412 (DMEM) with 1% antibiotics (Penicillin/Streptomycin (P/S)). The samples were mixed
413 using a bead beater (MP Biomedicals™ FastPrep-24™ 5G Bead Beating Grinder and Lysis System) at 4 m/s for 10 s, repeated three times with 90 s pauses on melting ice between each cycle. Homogenates were then clarified by centrifugation (2000 g, 10 min, 4°C).
414
415
416 Supernatants were collected, aliquoted and stored at -80°C until analysis. Viral RNA was then extracted from 200 µL of supernatant using the Maxwell® Viral TNA kit, according to the manufacturer's instructions (Promega, France). A negative control RNA extraction was performed for each set of 16 samples tested. All RNA extracts were stored at -80°C prior to use.
417
418
419
420

421 Virus production

422 SARS-CoV-2 Omicron BA.5 was gracefully obtained from Pasteur Institut - National
423 Reference Center for Viruses of Respiratory Infections (including Influenza and SARS-CoV-
424 2) (virus sequence GISAID accession number EPI_ISL_13017789). The virus was passaged
425 once on Calu-3 cells to increase the titer and the quantity available for animal infection
426 studies. The NGS virus sequence obtained after passage on Calu-3 cells was 100% similar
427 to the sequence of the virus stock.
428

428 Experimental design

429 Fifty-two wild rats with uncontrolled genetic background and microbiota were included
430 in this experimental study. This comprised twenty wild *Rattus rattus* (black rats) obtained
431 from a colony that has been maintained in an outdoor enclosure for over 20 years at the
432 veterinary school of Lyon (France). In the following, this group will be referred to as
433 *'Rattus rattus'*. Additionally, thirty-two wild *Rattus norvegicus* (brown rats) were
randomly selected from three distinct colonies A, B and C, each maintained in outdoor enclosures for over 20 years at the same facility. Animals from three different colonies were used in order to increase genetic diversity (the founder animals having been taken from different farms in the Lyon region). In the following, this group will be referred to as 'wild *Rattus norvegicus*. In addition to these wild specimens, forty laboratory rats with a known genetic background, consisting of twenty Sprague-Dawley strain and twenty Wistar strain, were included in the study. In the following, these groups will be referred to as 'Sprague-Dawley' and 'Wistar'. Rats were kept in cages with environmental enrichment and housed either individually (wild *Rattus norvegicus*) or in groups of two rats per cage. Negative animals were kept in separate room from the SARS-CoV-2 inoculated animals. Food and water were provided *ad libitum*. Weight of all animals were monitored and recorded on a daily basis throughout the duration of the experimental procedures.

## 447 Virus inoculation

Twenty-three wild *Rattus norvegicus*, fourteen wild *Rattus rattus*, fourteen Sprague-Dawley and fourteen Wistar were anaesthetized with isoflurane and intranasally inoculated (20µl in each nostril) with viral titters from $10^{4.57}$ TCID50 (50% Tissue Culture Infectious Dose)/40 µL to $10^{5.34}$ TCID50/40µL of SARS-CoV-2 Omicron BA.5 (Table 3). Nine wild *Rattus norvegicus*, six wild *Rattus rattus*, six Sprague-Dawley and six Wistar were inoculated with PBS in parallel to act as negative controls for each experimental protocol.
454 As described on Fig 8, SARS-CoV-2 and PBS inoculated rats were euthanised at either 4 or

455 14 days post-inoculation (dpi).

<table><caption>456 Table 3. Viral titers of Omicron BA.5 inoculated to the different rat groups</caption><thead><tr><th>Rat group</th><th>Viral titer inoculated of Omicron BA.5</th></tr></thead><tbody><tr><td>Rattus norvegicus (wild) – group A</td><td>10<sup>4.96</sup> TCID50/40 µL (10<sup>6.36</sup>TCDI50/mL)</td></tr><tr><td>Rattus norvegicus (wild) – group B</td><td>10<sup>5.34</sup> TCID50/40 µL (10<sup>6.74</sup>TCDI50/mL)</td></tr><tr><td>Rattus norvegicus (wild) – group C</td><td>10<sup>5.23</sup> TCID50/40 µL (10<sup>6.63</sup>TCDI50/mL)</td></tr><tr><td>Rattus norvegicus (wild)</td><td>10<sup>5.12</sup> TCID50/40 µL (10<sup>6.52</sup>TCDI50/mL)</td></tr><tr><td>Rattus rattus</td><td>10<sup>4.57</sup> TCID50/40 µL (10<sup>5.97</sup>TCDI50/mL)</td></tr><tr><td>Sprague-Dawley</td><td>10<sup>5.07</sup> TCID50/40 µL (10<sup>6.47</sup>TCDI50/mL)</td></tr><tr><td>Wistar</td><td>10<sup>5.18</sup> TCID50/40 µL (10<sup>6.58</sup>TCDI50/mL)</td></tr></tbody></table>

457 TCID50: 50% Tissue Culture Infectious Dose

458

<caption>459 Fig 8. Experimental design including four groups of rats intranasally inoculated with Omicron BA.5.</caption>

460 Wild *Rattus norvegicus* (blue), wild *Rattus rattus* (green), Sprague-Dawley laboratory rats (orange) and
461 Wistar laboratory rats (purple), each inoculated with either SARS-CoV-2 Omicron BA.5 or PBS (negative
462 control groups (black)) were necropsied at 4 (D4) or 14 (D14) days post infection.

463 **In vivo experiments and samples**

464 Blood was collected by cardiac puncture under deep anaesthesia (Isoflurane, Ketamine
465 (100 mg/kg) and Xylazine (10 mg/kg)) prior to euthanasia. Blood was collected into 4 mL
466 K3-EDTA tubes. Following euthanasia, all animals were necropsied to collect lungs and
467 upper respiratory tract (URT). After centrifugation of blood samples (1000 g/15 min),
468 plasmas were stored at -20°C until analysis. For viral RNA extraction, lungs or URT tissues

469 (~20 to 60 mg) were prepared as described previously (section rat captures and samples).

470 Viral RNA was extracted from 160 µL of previously obtained homogenate using the Qiagen

471 Viral RNA mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer's

472 instructions. All RNA extracts were stored at -80°C before use.

473 **SARS-CoV-2 viral titrations on cell culture**

474 SARS-CoV-2 titrations were performed on lung and URT samples by TCID50 assays on Vero

475 E6 cells.

476 Briefly, 96-well plates were seeded with a Vero E6 cell suspension (1.10⁵ cells/mL) 24 h

477 before virus inoculation. A ten-fold serial dilution of tissue homogenates and the

478 reference virus (used as positive control) was performed in DMEM supplemented with

479 10% Foetal Calf Serum (FCS) and 1% antibiotics (e.g. mix of P/S). After removing the cell

480 culture medium, inoculum was added to each well and plates were incubated at 37°C in

481 5% CO₂ for 1 h. Six uninfected wells were used as independent negative controls. After

482 the 1 h incubation, 200 µL of DMEM supplemented with 10% FCS and 1% mix of P/S were

483 added to each well and the plates were incubated at 37°C in 5% CO₂ for 72 h. Viral titers

484 were calculated in TCID50/mL using the Spearman-Kärber method.

485 In addition, viral inoculums were titrated on cells both before and after rat inoculations.

486 **Microsphere immunoassay (Luminex Technology)**

487 For plasma samples from experimental *in vivo* studies and sera from field studies, anti-

488 SARS-CoV-2 antibodies were detected by microsphere immunoassay (MIA) as described
489 by Bourret et al. [33]. Three recombinant SARS-CoV-2 antigens (nucleoprotein, spike
490 subunit 1 receptor binding domain (RBD) and trimeric spike (its native form) (The Native
491 Antigen Company)) were used to capture specific plasma/serum antibodies. BSA (Bovine
492 Serum Albumin) was used as a control antigen. Plasma samples from in vivo studies were
493 previously diluted 1:20 while sera from field studies were diluted 1:10 in assay buffer
494 (PBS-1% BSA-0.05% Tween 20, (Sigma-Aldrich)). The assay was performed on a Bio-Plex
495 200 instrument (Bio-Rad). A minimum of 100 events were read for each bead set and
496 binding events were expressed as median fluorescence intensity (MFI). Relative
497 Fluorescence Intensity (RFI) was calculated for each sample by dividing the MFI signal
498 measured for the antigen coated microsphere sets by the MFI signal obtained for the
499 control microsphere set (BSA coated beads), to account for non-specific binding of
500 antibodies to the beads. Specific seropositivity cut-off values for each antigen were set at
501 three standard deviations above the mean RFI of negative samples. To determine the
502 seropositivity cut-off for the field samples, given the low prevalence of SARS-CoV-2 in wild
503 rodents, the entire rat population (mean of all RFIs + 3 standard deviation) was used as
504 described by Fritz et al. [21]. Seroconversion was defined as the detection of antibodies
505 to the three antigens and inconclusive result was defined as the detection of antibodies
506 to two of the three antigens.

507 Seroneutralisation assay

508 Seroneutralisation assays were carried out on heat-inactivated plasmas/sera (56°C/30
509 min) collected from all rats included in the in vivo experiments, as well as on
510 positive/inconclusive sera by MIA collected from urban rodents. The SARS-CoV-2 Omicron
511 BA.5 (virus sequence GISAID accession number EPI_ISL_13017789) and the SARS-CoV-2
512 D614G (virus sequence GISAID accession number EPI_ISL_666870) variants were used
513 respectively as challenge viruses as described by Bourret et al. [33]. Briefly, Vero E6 cells
514 were seeded in 96-well plates at 1.10⁵ cells/mL. One day later, plasmas, positive and
515 negative controls were serially diluted in a 1 to 3 dilution steps in cell culture medium
516 (DMEM supplemented with 10% SVF and 1% P/S) and 50 µL of virus previously diluted to
517 100 TCID50/50µL were added to samples and controls in the 96-well plates. The plates
518 were incubated at 37°C (5% CO₂) for 1 h. Afterwards, the cell culture supernatants were
519 removed and replaced with 100 µL of the mix of virus and serially diluted samples or
520 controls. After at least 3 days of incubation, the presence of viral cytopathic effect (CPE)
521 was quantified by an “all or nothing” reading method. The neutralisation titer was
522 calculated from the highest dilution that prevented detectable cytopathic effect.

523 TaqMan RT-qPCR

524 TaqMan RT-qPCR targeting the envelope protein gene (E gene) was performed using the
525 Quantitect Probe RT-PCR kit (Qiagen, Courtaboeuf, France). The model of primers/probe
526 used was as follows: primer E_Sarbeco_F (forward): 5'-
527 ACAGGTACGTTAATAGTTAATAGCGT, primer E_Sarbeco_R (reverse): 5'-
528 ATATTGCAGCAGTACGCACACA and the probe E_Sarbeco_P1 5'-FAM-
529 ACACTAGCCATCCTTA CTGCGCTTCG-BHQ-1. Primers and probe were supplied by
530 Eurogentec (Angers, France).
531 PCR was performed as previously described in Monchatre Leroy *et al.* with minor modifications [43]. TaqMan RT-qPCR was performed in a total volume of 20 µL containing 532 2.5 µL of RNA sample, 10 µL of 2x Master Mix, 5.3 µL of RNase-free water, 0.8 µL each of 533 forward and reverse primer (10 µM), 0.4 µL of probe (10 µM), and 0.2 µL of QuantiTect 534 RT Mix. All TaqMan RT-qPCR assays were performed on the thermocycler Rotor Gene Q 535 MDx (Qiagen, Courtaboeuf, France). Amplification was performed under the following 536 thermocycling conditions: 50°C for 30 min for reverse transcription, followed by 95°C for 537 15 min and then 45 cycles of 94°C for 15 s and 60°C for 60 s. Negative and positive controls 538 were included in each RT-qPCR assay. 539

540 Copy number/µL in each sample was determined using six 10-fold serial dilutions of SARS 541 COV-2 RNA at 3.23x10⁸ copies/µL (3.23x10⁷ - 3.23x10²). A cut-off of 0.03 was used as the 542 reference threshold for each RT-qPCR assay. Efficiency, slope and correlation coefficient 543 (R²) were calculated directly by the Rotor Gene software. All reactions were performed in 544 duplicates. 545

545 Finally, the PCR detection limit was determined by testing five serial dilutions of SARS- 546 COV-2 from 500 to 5 copies/µL, each dilution being performed in quadruplicate (i.e. 12 547 replicates per dilution). LDₚ₄R was demonstrated > 25 copies/µL (last dilution giving 100% 548 positive replicates). 549

549 SARS-CoV-2 RT-qPCR, targeting the polymerase gene, was performed on purified nucleic 550 acids extracted from sewage collected in sewers as described in [44] for Nancy, Besançon, 551 Nantes and Lyon where rodent were trapped directly in sewers.
552 Molecular modelling procedures

553 The complete structures (including the transmembrane domain) of *Homo sapiens* and
554 *Rattus norvegicus* ACE2 receptors were obtained from AlphaFold protein structure
555 database [45,46] and can be found using their UniProt identifiers (Q9BYF1 and Q5EGZ1,
556 respectively). The structural model of the *Rattus rattus* ACE2 receptor was obtained using
557 AlphaFold Colab notebook [47] based on the GenBank sequence (XM_032890254.1). The
558 Receptor Binding Domain (RBD) structure of the Omicron BA.5 Spike protein was obtained
559 from the Protein Data Bank [48], PDB ID: 7XWA [49]. ACE2-RBD complexes were finally
560 built by superimposing their respective structures on the crystallographic structure of the
561 above complex (7XWA), using PyMOL software (PyMOL).

562 Molecular dynamics (MD) systems preparation and simulations were performed using the
563 GROMACS 2018 package software [50], with the CHARMM-36 forcefield applied [51].
564 Each ACE2-RBD complex was first embedded into a previously equilibrated
565 phosphatidylcholine (DLPC) membrane [52] using the *membed* function of the GROMACS
566 software. Solvation, energy minimization, 100-ps temperature and 1-ns pressure
567 equilibrations were carried out using GROMACS routines (V-rescale thermostat,
568 Berendsen pressure coupling, and semi-isotropic coupling type were applied). A 500-ns
569 MD simulation was performed on each ACE2-RBD complex to equilibrate the systems. The
570 *cluster* function of GROMACS, with the *gromos* algorithm and a 4 Å cut-off, was then
571 applied to identify the most representative conformation of each complex, finally used
572 for Potential of Mean Force (PMF) calculations.
573 PMF calculations involved ACE2 and RBD as pull groups (positions of the ACE2 receptors

574 were restrained), which defined the Z-axis as the reaction coordinate. A harmonic

575 potential and a distance increase over this reaction coordinate were then applied, with a

576 constant velocity of 100 Å/ns and a force constant of 1000 kJ/mol/nm². After this pull

577 step, the RBD positions along the reaction coordinate were sampled every 2 Å, until the

578 RBD center of mass reached a 60 Å distance from the ACE2 receptor centre of mass. The

579 Umbrella Sampling (US) step was then performed on the 11 resulting conformations, with

580 a 1000 kJ/nm/nm² constant force over 4-ns simulations. Finally, the PMF values were

581 extracted using the weighted histogram analysis method implemented in GROMACS.

582 Percentages of identity between ACE2 sequences were calculated using Geneious Prime®

583 2022.0.2 version.

584 Statistical analyses and graphs

585 Graphs and statistical analyses (Kruskall-Wallis and one-way ANOVA) were performed

586 with GraphPad Prism (version 9) and PyMOL (version 4.6.0). A p-value of <0.05 was

587 considered statistically significant. Figs 1 and 8 were generated using images from Servier

588 Medical Art by Servier, licensed under a Creative Commons Attribution 4.0 Unported

589 License (https://creativecommons.org/licenses/by/4.0/).

590
591 Acknowledgements

592 We would like to thank all the partners from urban cities involved in the pest control

593 programs, the teams of ANSES Laboratory for Rabies and Wildlife including Virologie

594 Moléculaire (C. Peytavin De Garam, F. Bastien and J.L. Schereffer) and Virologie-

595 Immunologie-Sérologie (J. Rieder, A. Labadie, F. Chanteclair and J. Bonetti) and Service

596 d'Expérimentation Animale Rongeurs (S. Kempff, V. Brogat, and E. Litaize) and Service

597 d'Expérimentation Animale Carnivores (M. Vesvres). We would like to thank Sylvie Van

598 der Werf and Flora Donati from Pasteur Institut - National Reference Center for Viruses

599 of Respiratory Infections (including Influenza and SARS-CoV-2) for providing SARS-CoV-2

600 Omicron BA.5.

601 Funding

602 This work was funded by l'ANRS/Santé Publique France - Projet EMERGEN (ANRS0163).

603 This work was granted access to the HPC resources of IDRIS under the allocation

604 AD010715471 made by GENCI.

605 References

606 1. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak

607 associated with a new coronavirus of probable bat origin. Nature.

608 2020;579(7798):270-3.

609 2. Qiu X, Liu Y, Sha A. SARS-CoV-2 and natural infection in animals. J Med Virol. 2022 Sep

610 28;10.1002/jmv.28147.
611 3. Sailleau C, Dumarest M, Vanhomwegen J, Delaplace M, Caro V, Kwasiborski A, et al.
612 First detection and genome sequencing of SARS-CoV-2 in an infected cat in France.
613 Transbound Emerg Dis. 2020 Jun 5;10.1111/tbed.13659.

614 4. Sit TH, Brackman CJ, Ip SM, Tam KW, Law PY, To EM, et al. Canine SARS-CoV-2
615 infection. Nature. 2020 Oct 5;586(7831):776-8.

616 5. Oreshkova N, Molenaar RJ, Vreman S, Harders F, Oude Munnink BB, Hakze-van der
617 Honing RW, et al. SARS-CoV-2 infection in farmed minks, the Netherlands, April and
618 May 2020. Euro Surveill Bull Eur Sur Mal Transm Eur Commun Dis Bull. 2020
619 Jun;25(23):2001005.

620 6. McAloose D, Laverack M, Wang L, Killian ML, Caserta LC, Yuan F, et al. From People to
621 Panthera: Natural SARS-CoV-2 Infection in Tigers and Lions at the Bronx Zoo. mBio.
622 2020 Oct 13;11(5):e02220-20.

623 7. Padilla-Blanco M, Aguiló-Gisbert J, Rubio V, Lizana V, Chillida-Martínez E, Cardells J,
624 et al. The Finding of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-
625 2) in a Wild Eurasian River Otter (Lutra lutra) Highlights the Need for Viral Surveillance
626 in Wild Mustelids. Front Vet Sci. 2022 Mar 31;9:826991.

627 8. Kuchipudi SV, Surendran-Nair M, Ruden RM, Yon M, Nissly RH, Vandegrift KJ, et al.
628 Multiple spillovers from humans and onward transmission of SARS-CoV-2 in white-
629 tailed deer. Proc Natl Acad Sci U S A. 2022 Feb 8;119(6):e2121644119.

630 9. Larsen HD, Fonager J, Lomholt FK, Dalby T, Benedetti G, Kristensen B, et al.
631 Preliminary report of an outbreak of SARS-CoV-2 in mink and mink farmers associated
632 with community spread, Denmark, June to November 2020. Euro Surveill Bull Eur Sur
633 Mal Transm Eur Commun Dis Bull. 2021 Feb;26(5):2100009.

634 10. Pickering B, Lung O, Maguire F, Kruczkiewicz P, Kotwa JD, Buchanan T, et al. Divergent
635 SARS-CoV-2 variant emerges in white-tailed deer with deer-to-human transmission.
636 Nat Microbiol. 2022 Dec;7(12):2011-24.

637 11. Bao L, Deng W, Huang B, Gao H, Liu J, Ren L, et al. The pathogenicity of SARS-CoV-2 in
638 hACE2 transgenic mice. Nature. 2020 Jul;583(7818):830-3.

639 12. Shuai H, Chan JFW, Yuen TTT, Yoon C, Hu JC, Wen L, et al. Emerging SARS-CoV-2
640 variants expand species tropism to murines. EBioMedicine. 2021 Nov;73:103643.

641 13. Muñoz-Fontela C, Dowling WE, Funnell SGP, Gsell PS, Balta XR, Albrecht RA, et al.
642 Animal models for COVID-19. Nature. 2020 Oct 5;586(7830):509-15.

643 14. Zhang C, Cui H, Li E, Guo Z, Wang T, Yan F, et al. The SARS-CoV-2 B.1.351 Variant Can
644 Transmit in Rats But Not in Mice. Front Immunol. 2022;13:869809.
645 15. Wang Y, Lenoch J, Kohler D, DeLiberto TJ, Tang CY, Li T, et al. SARS-CoV-2 Exposure in Norway Rats (Rattus norvegicus) from New York City. mBio. 2023 Apr 646 25;14(2):e0362122.
647

648 16. Montagutelli X, Prot M, Levillayer L, Salazar EB, Jouvion G, Conquet L, et al. Variants with the N501Y mutation extend SARS-CoV-2 host range to mice, with contact transmission. BioRxiv [Preprint]. 2021 ;2021.03.18.436013 [posted 2021 Dec 07; cited 650 2024 Dec 10]: [22 p]. Available from: 651 652 https://www.biorxiv.org/content/10.1101/2021.03.18.436013v2 doi: 653 10.1101/2021.03.18.436013

654 17. Smyth DS, Trujillo M, Gregory DA, Cheung K, Gao A, Graham M, et al. Tracking cryptic 655 SARS-CoV-2 lineages detected in NYC wastewater. Nat Commun. 2022 Feb 656 3;13(1):635.

657 18. Sherchan S, Thakali O, Ikner LA, Gerba CP. Survival of SARS-CoV-2 in wastewater. Sci 658 Total Environ. 2023 Jul 15;882:163049.

659 19. Fisher AM, Airey G, Liu Y, Gemmell M, Thomas J, Bentley EG, et al. The ecology of 660 viruses in urban rodents with a focus on SARS-CoV-2. Emerg Microbes Infect. 2023 661 Dec 31;12(1):2217940.

662 20. Robinson SJ, Kotwa JD, Jeeves SP, Himsworth CG, Pearl DL, Weese JS, et al. 663 Surveillance for SARS-CoV-2 in Norway Rats (Rattus norvegicus) from Southern 664 Ontario. Wang L, editor. Transbound Emerg Dis. 2023 May 26;2023:1-9.

665 21. Fritz M, de Riols de Fonclare D, Garcia D, Beurlet S, Becquart P, Rosolen SG, et al. First 666 Evidence of Natural SARS-CoV-2 Infection in Domestic Rabbits. Vet Sci. 2022 Jan 667 27;9(2):49.

668 22. Coronavirus : circulation des variants du SARS-CoV-2 [Internet]. [cited 2024 Dec 10]. 669 Available from: https://www.santepubliquefrance.fr/dossiers/coronavirus-covid- 670 19/coronavirus-circulation-des-variants-du-sars-cov-2

671 23. Wurtzer S, Waldman P, Levert M, Cluzel N, Almayrac JL, Charpentier C, et al. SARS- 672 CoV-2 genome quantification in wastewaters at regional and city scale allows precise 673 monitoring of the whole outbreaks dynamics and variants spreading in the 674 population. Sci Total Environ. 2022 Mar 1;810:152213.

675 24. Colombo VC, Sluydts V, Mariën J, Vanden Broecke B, Van Houtte N, Leirs W, et al. 676 SARS-CoV-2 surveillance in Norway rats (Rattus norvegicus) from Antwerp sewer 677 system, Belgium. Transbound Emerg Dis. 2021 Jul 10;10.1111/tbed.14219.

678 25. Wernike K, Drewes S, Mehl C, Hesse C, Imholt C, Jacob J, et al. No Evidence for the 679 Presence of SARS-CoV-2 in Bank Voles and Other Rodents in Germany, 2020-2022. 680 Pathogens. 2022 Sep 28;11(10):1112.
681 26. Fernández-Bastit L, Montalvo T, Franco S, Barahona L, López-Bejar M, Carbajal A, et al. Monitoring SARS-CoV-2 infection in urban and peri-urban wildlife species from Catalonia (Spain). One Health Outlook. 2024 Sep 1;6(1):15.

684 27. Lee LKF, Himsworth CG, Prystajecky N, Dibernardo A, Lindsay LR, Albers TM, et al. SARS-CoV-2 Surveillance of Wild Mice and Rats in North American Cities. EcoHealth. 2024 Mar;21(1):1-8.

687 28. Miot EF, Worthington BM, Ng KH, Lataillade L de G de, Pierce MP, Liao Y, et al. Surveillance of Rodent Pests for SARS-CoV-2 and Other Coronaviruses, Hong Kong. Emerg Infect Dis J - CDC. 2022 Feb 1;28(2):467-470.

690 29. Martínez-Hernández F, Gonzalez-Arenas NR, Cervantes JAO, Villalobos G, Olivo-Diaz A, Rendon-Franco E, et al. Identification of SARS-CoV-2 in urban rodents from Southern Mexico City at the beginning of the COVID-19 pandemic. Rev Inst Med Trop 2024 Feb 5;66:e8.

694 30. Robinson CA, Hsieh HY, Hsu SY, Wang Y, Salcedo BT, Belenchia A, et al. Defining biological and biophysical properties of SARS-CoV-2 genetic material in wastewater. Sci Total Environ. 2022 Feb 10;807(Pt 1):150786.

697 31. Halfmann PJ, Iida S, Iwatsuki-Horimoto K, Maemura T, Kiso M, Scheaffer SM, et al. SARS-CoV-2 Omicron virus causes attenuated disease in mice and hamsters. Nature. 2022;603(7902):687-92.

700 32. Shuai H, Chan JFW, Hu B, Chai Y, Yuen TTT, Yin F, et al. Attenuated replication and pathogenicity of SARS-CoV-2 B.1.1.529 Omicron. Nature. 2022 Mar;603(7902):693-9.

702 33. Bourret V, Dutra L, Alburkat H, Mäki S, Lintunen E, Wasniewski M, et al. Serologic Surveillance for SARS-CoV-2 Infection among Wild Rodents, Europe. Emerg Infect Dis 2024;28(12):2577-80.

705 34. Wernike K, Mehl C, Aebischer A, Ulrich L, Heising M, Ulrich RG, et al. SARS-CoV-2 and Other Coronaviruses in Rats, Berlin, Germany, 2023. Emerg Infect Dis. 2024 Oct;30(10):2205-8.

708 35. Tan CS, Adrus M, Rahman SPH, Azman HIM, Abang RAA. Seroevidence of SARS-CoV-2 spillback to rodents in Sarawak, Malaysian Borneo. BMC Vet Res. 2024 Apr 27;20(1):161.

711 36. Perera RAPM, Ko R, Tsang OTY, Hui DSC, Kwan MYM, Brackman CJ, et al. Evaluation of a SARS-CoV-2 Surrogate Virus Neutralization Test for Detection of Antibody in Human, Canine, Cat, and Hamster Sera. J Clin Microbiol. 2021 Jan 21;59(2):e02504-20.
715 37. Mariën J, Michiels J, Heyndrickx L, Nkuba-Ndaye A, Ceulemans A, Bartholomeeusen
716 K, et al. Evaluation of a surrogate virus neutralization test for high-throughput
717 serosurveillance of SARS-CoV-2. J Virol Methods. 2021 Nov;297:114228.

718 38. Hulst M, Kant A, Harders-Westerveen J, Hoffmann M, Xie Y, Laheij C, et al. Cross-
719 Reactivity of Human, Wild Boar, and Farm Animal Sera from Pre- and Post-Pandemic
720 Periods with Alpha- and Beta-Coronaviruses (CoV), including SARS-CoV-2. Viruses.
721 2023 Dec 23;16(1):34.

722 39. Lau SKP, Woo PCY, Li KSM, Tsang AKL, Fan RYY, Luk HKH, et al. Discovery of a Novel
723 Coronavirus, China Rattus Coronavirus HKU24, from Norway Rats Supports the
724 Murine Origin of Betacoronavirus 1 and Has Implications for the Ancestor of
725 Betacoronavirus Lineage A. J Virol. 2014 Dec 31;89(6):3076-92.

726 40. Viney M, Riley EM. The Immunology of Wild Rodents: Current Status and Future
727 Prospects. Front Immunol. 2017;8:1481.

728 41. Directive 2010/63/EU of the European Parliament and of the Council of 22 September
729 2010 on the protection of animals used for scientific purposes Text with EEA relevance
730 [Internet]. 2010 [cited 2024 Dec 10]. Available from:
731 http://data.europa.eu/eli/dir/2010/63/oj/eng

732 42. Article 1 - Décret n° 2013-118 du 1er février 2013 relatif à la protection des animaux
733 utilisés à des fins scientifiques - Légifrance [Internet]. [cited 2024 Dec 10]. Available
734 from: https://www.legifrance.gouv.fr/jorf/article_jo/JORFARTI000027037854

735 43. Monchatre-Leroy E, Lesellier S, Wasniewski M, Picard-Meyer E, Richomme C, Boué F,
736 et al. Hamster and ferret experimental infection with intranasal low dose of a single
737 strain of SARS-CoV-2. J Gen Virol. 2021 Mar;102(3):001567.

738 44. Vincent-Hubert F, Wacrenier C, Desdouits M, Jousse S, Schaeffer J, Le Mehaute P, et
739 al. Development of passive samplers for the detection of SARS-CoV-2 in sewage and
740 seawater: Application for the monitoring of sewage. Sci Total Environ. 2022 Aug
741 10;833:155139.

742 45. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly
743 accurate protein structure prediction with AlphaFold. Nature. 2021
744 Aug;596(7873):583-9.

745 46. Varadi M, Bertoni D, Magana P, Paramval U, Pidruchna I, Radhakrishnan M, et al.
746 AlphaFold Protein Structure Database in 2024: providing structure coverage for over
747 214 million protein sequences. Nucleic Acids Res. 2024 Jan 5;52(D1):D368-75.

748 47. Mirdita M, Steinegger M, Söding J. MMseqs2 desktop and local web server app for
749 fast, interactive sequence searches. Bioinformatics. 2019 Aug 15;35(16):2856-8.
750 48. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The Protein Data Bank. Nucleic Acids Res. 2000 Jan 1;28(1):235-42.

752 49. Kimura I, Yamasoba D, Tamura T, Nao N, Suzuki T, Oda Y, et al. Virological characteristics of the SARS-CoV-2 Omicron BA.2 subvariants, including BA.4 and BA.5. Cell. 2022 Oct;185(21):3992-4007.e16.

755 50. Lemkul JA. From Proteins to Perturbed Hamiltonians: A Suite of Tutorials for the GROMACS-2018 Molecular Simulation Package [Article v1.0]. Living J Comput Mol Sci. 2019;1(1):5068-5068.

758 51. Best RB, Zhu X, Shim J, Lopes PEM, Mittal J, Feig M, et al. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ(1) and χ(2) dihedral angles. J Chem Theory Comput. 2012 Sep 11;8(9):3257-73.

762 52. Jämbeck JPM, Lyubartsev AP. Derivation and systematic validation of a refined all-atom force field for phosphatidylcholine lipids. J Phys Chem B. 2012 Mar 15;116(10):3164-79.

765

766 Supporting information

767 S1 Fig. Alignments of *Homo sapiens*, *Rattus norvegicus* and *Rattus rattus* ACE2.

768

769
<figure><img src="image_2.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2024.12.19.629569; this version posted December 20, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

<caption>Fig1</caption>
<figure><img src="image_3.png" /><figcaption>Fig2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig3</figcaption></figure>
RNA copies/µl of supernatant

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2024.04.12.19629569; this version posted December 20, 2024. The copyright holder for this preprint (which has not been peer reviewed) may not permit its use, distribution or republication, without permission. In most cases, permission to reuse a preprint should be sought directly from the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

Negative

Rattus norvegicus (wild)

Rattus rattus

Sprague-Dawley

Wistar
A

Rattus norvegicus

<figure><img src="image_16.png" /></figure>

B

Rattus rattus

<figure><img src="image_17.png" /></figure>

C

Sprague-Dawley

<figure><img src="image_18.png" /></figure>

D

Wistar

<figure><img src="image_19.png" /></figure>
Neutralising antibodies

bioRxiv preprint doi: https://doi.org/10.1101/2024.12.19.629569; this version posted December 20, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

<figure><img src="image_20.png" /></figure>
A

<figure><img src="image_21.png" /></figure>

B

<figure><img src="image_22.png" /></figure>

C

<figure><img src="image_23.png" /></figure>

D

ACE2-RBD affinity

<figure><img src="image_24.png" /></figure>
<figure><img src="image_25.png" /></figure>

Wild rats

1 *Rattus norvegicus*

2 *Rattus rattus*

N=Omicron BA.5

N=Negative

D4†

D14†

N=15

N=6

N=8

N=3

N=7

N=3

N=7

N=3

Laboratory
rats

3 Sprague-Dawley

4 Wistar

N=7

N=3

N=7

N=3

N=7

N=3

N=7

N=3

†Necropsy