key: cord-280392-ij5gtesw
authors: Gultom, Mitra; Licheri, Matthias; Laloli, Laura; Wider, Manon; Strässle, Marina; Steiner, Silvio; Kratzel, Annika; Thao, Tran Thi Nhu; Stalder, Hanspeter; Portmann, Jasmine; Holwerda, Melle; V’kovski, Philip; Ebert, Nadine; Stokar – Regenscheit, Nadine; Gurtner, Corinne; Zanolari, Patrik; Posthaus, Horst; Schuller, Simone; Vicente – Santos, Amanda; Moreira – Soto, Andres; Corrales – Aguilar, Eugenia; Ruggli, Nicolas; Tekes, Gergely; von Messling, Veronika; Sawatsky, Bevan; Thiel, Volker; Dijkman, Ronald
title: Susceptibility of well-differentiated airway epithelial cell cultures from domestic and wildlife animals to SARS-CoV-2
date: 2020-11-10
journal: bioRxiv
DOI: 10.1101/2020.11.10.374587
sha: 
doc_id: 280392
cord_uid: ij5gtesw

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally, and the number of cases continues to rise all over the world. Besides humans, the zoonotic origin, as well as intermediate and potential spillback host reservoirs of SARS-CoV-2 are unknown. To circumvent ethical and experimental constraints, and more importantly, to reduce and refine animal experimentation, we employed our airway epithelial cell (AEC) culture repository composed of various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. In this study, we inoculated well-differentiated animal AEC cultures of monkey, cat, ferret, dog, rabbit, pig, cattle, goat, llama, camel, and two neotropical bat species with SARS-CoV-2. We observed that SARS-CoV-2 only replicated efficiently in monkey and cat AEC culture models. Whole-genome sequencing of progeny virus revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat epithelial airway cells. Our findings, together with the previously reported human-to-animal spillover events warrants close surveillance to understand the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

Introduction animals studied, only monkey and cat airway epithelial cells support efficient replication of SARS-CoV-111

The absence of infectious progeny virus in most animal species, except rhesus macaques and 113 cats, indicates that certain animal species may be intrinsically refractory to SARS-CoV-2 infection, 114 which may be due to incompatibility with the cellular receptor utilized by SARS-CoV-2 for cellular entry 115 25, 26 . To assess whether the observed susceptibility to SARS-CoV-2 corresponds to the amino acid 116 sequence conservation of the receptor-binding motif (RBM) in ACE2 we performed in silico analysis on 117 the available ACE2 protein sequences 25, 27 . The ACE2 protein sequences from the two neotropical bat 118 species (S. lilium and C. perspicillata) were not included in the analysis, due to their unavailability. 119

Similarly, the ACE2 protein sequence for llama is not available, and therefore we used the sequence of 120 alpaca (XM_006212647.3) as an alternative, as it is the closest relative. This revealed that in 121 comparison to humans, the ACE2 RBM regions interacting with the receptor-binding domain (RBD) of 122 SARS-CoV-2 are well conserved in rhesus macaques and cats while being slightly more diverse in other 123 species (Fig. S2a) . Orthomyxoviridae and are known to have a broad host spectrum, including ferrets [15] [16] [17] 29 . The AEC 131 cultures from 12 different species (rhesus macaque, cat, ferret, dog, rabbit, pig, cattle, goat, llama, 132 camel, and two neotropical bats) were inoculated with 10.000 TCID50 of either IAV or IDV and incubated 133 at 33°C and 37°C. After 48 hours, the AEC cultures were fixed and processed by immunofluorescence 134 assays. This analysis showed that, in contrast to SARS-CoV-2, IAV antigen-positive cells could be 135 detected in both companion animals AEC cultures, as well as in the commonly used animal models, 136 such as ferret, monkey, rabbit, and porcine AEC cultures (Fig. 2, Fig. S1a ) 30 . For IDV we observed 137 antigen-positive cells in all AEC model, except for rhesus macaque and one of the neotropical bat 138 species, indicating that the AEC cultures were all well-differentiated and susceptible to virus infection. 139

In the immunofluorescence analysis we also incorporated an antibody against beta-tubulin 140 marker to discern ciliated and non-ciliated cell populations. For both rhesus macaques and cats, SARS-141

CoV-2 antigen-positive cells predominantly overlapped with the non-ciliated cell populations, 142

irrespective of the incubation temperature. Using a polyclonal antibody against the human ACE2 we 143 could observe that the cellular receptor expression in rhesus macaques predominantly overlaps with 144 SARS-CoV-2 cell tropism, indicating same cellular tropism (Fig. S2b) . Unfortunately, the polyclonal antibody against the human ACE2 did not bind the feline ACE2 protein, we could therefore not formally 146 demonstrate that SARS-CoV-2 virus-infected cat cells are indeed expressing ACE2 on their surface. 147 148

It has previously been shown that SARS-CoV-2 can undergo rapid genetic changes in vitro 31 . Since we 150 observed efficient replication in rhesus macaque and cat AEC cultures, therefore we assessed whether 151 any mutations suggestive of viral adaptation had occurred. We performed whole-genome sequencing 152 (Nanopore sequencing technology) on the viral inoculum used as well as the progeny viruses collected 153 after one passage, at 96 hpi from the rhesus macaque and cat AEC cultures incubated at 33°C and 37°C. 154

This inoculum was either passage 1 or passage 2 virus stocks from the SARS-CoV-2/München-155 1.1/2020/929 isolate we had received. In the viral sequences in the 96 hpi samples from virus-infected 156 rhesus macaque and cat AEC cultures, we observed no obvious signs of nucleotide transitions that lead 157 to nonsynonymous mutations compared to the respective inoculums ( Fig. 3) , irrespective of 158 temperature and animal species. This highlights that the currently circulating SARS-CoV-2 D614G-159 variant can productively infect rhesus macaque and cat airway epithelial cells. sequencing indicated that the current circulating SARS-CoV-2 D614G-variant can efficiently infect 167 rhesus macaque and cat airway epithelial cells. Our data highlight that these two animals are potential 168 models for the evaluation of therapeutic mitigation strategies for currently circulating viral variants. In 169 conjunction with the previous documented spillover events, close surveillance of these animals, 170

including closely related species, in the wild, captivity, and household situations is warranted. 171

To date, there have been several published reports evaluating the suitability of animal models 172 towards SARS-CoV-2 infection, including cats, rhesus macaques, dogs, pigs, and ferrets 22,23,32-34 . 173

Interestingly, we observed that SARS-CoV-2 does not efficiently replicate in our tracheobronchial 174 airway epithelial cells derived from ferrets, whereas ferrets are used as animal models. This may be 175 due to viral infections in ferrets are mainly restricted to the nasal conchae and are dose-dependent, 176 and additionally, the origin of the cells used as input for the AEC may not recapitulate the cells of the 177 nasal mucosa 24,32,34 . It is known that there are differences in cellular composition and the host 178 determinant expression levels along proximal and distal regions of the respiratory tract 35 . Additionally, SARS-CoV-2 may even utilize a different cellular receptor in ferrets 36 . Therefore, it would be of interest 180 to complement our current repository with AEC cultures from different anatomical regions of animals 181 like ferrets and to evaluate whether ACE2 is employed by SARS-CoV-2 as the cellular receptor in the 182 different animal species. 183

It has been proposed that SARS-CoV-2 spillover into the human population, like SARS-CoV, has 184 originated from bats, either directly or via an intermediate reservoir 3, 37 . With more than 1200 bat 185 species comprising more than 20% of all mammalian species, we restricted our experiments with SARS-186

CoV-2 to our established AEC cultures from the two neotropical Carollia perspicillata and Sturnira lilium 187 bat species (Gultom et al Manuscript in preparation) . We show that these two neotropical bats are not 188 susceptible to SARS-CoV-2, suggesting that they are not a likely reservoir host for SARS-CoV-2, despite 189 the detection of other coronaviruses and presumptive ACE2 receptor usage by SARS-CoV-2 in the 190 closely related bat species 38,39 . Interestingly, it has recently been described that fruit bats (Rousettus and stored at -80°C for later analysis. Following the collection of the apical washes, the basolateral 246 medium was exchanged with fresh ALI medium. Each experiment was repeated as two independent 247 biological replicates using AEC cultures established from either one or two biological donors of each 248 species, depending on the availability of procured animal tissue (Table. 1 

For the quantification of SARS-CoV-2 apical washes were titrated by plaque assay on Vero E6 cells. 285

Briefly, 1x10 5 cells/well were seeded in 24-well plates one day prior to the titration and inoculated 286 with 10-fold serial dilutions of virus solutions. Inoculums were removed 1 hpi and replaced with 287 overlay medium consisting of DMEM supplemented with 1.2% Avicel (RC-581, FMC biopolymer), 10% 288 heat-inactivated FBS, 100 µg/ml streptomycin, and 100 IU/ml penicillin. Cells were incubated at 37 °C 289 with 5% CO2 for 48 hours and fixed with 4% (v/v) neutral buffered formalin prior to staining with crystal 290 violet 47 . 291

For the analysis on the conservation of ACE2 among different species, the available ACE2 protein ClustalW in Geneious 11.1.5. Software (Biomatters) using the default setting. ACE2 protein residues 298 interacting with SARS-CoV-2, receptor binding motif (RBM), were selected based on previous described 299 critical ACE2 residues interacting with SARS-CoV-2 receptor binding domain (RBD) 25, 27 . 300

Sequencing was performed on viral RNA isolated from the SARS-CoV-2 stock and the 96 hpi apical 302 washes of SARS-CoV-2-infected monkey and cat AEC cultures according to the ARTIC platform nCoV19 303 protocols 48,49 . The v2 protocol was used as a basis for the reverse transcript and tiled multiplex PCR 304 reaction using the ARTIC nCoV-2019 V3 primer pool (see Table. 

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