key: cord-0255044-9chl1nci
authors: Kapczynski, Darrell R.; Sweeney, Ryan; Suarez, David L.; Spackman, Erica; Pantin-Jackwood, Mary
title: Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells
date: 2021-08-19
journal: bioRxiv
DOI: 10.1101/2021.08.18.456916
sha: 15c3d0e8ac612b3dcc4eec2797e317c3a9e4d84d
doc_id: 255044
cord_uid: 9chl1nci

The SARS-CoV-2 (SC2) virus has caused a worldwide pandemic because of the virus’s ability to transmit efficiently human-to-human. A key determinant of infection is the attachment of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Because of the presumed zoonotic origin of SC2, there is no practical way to assess every species susceptibility to SC2 by direct challenge studies. In an effort to have a better predictive model of animal host susceptibility to SC2, we expressed the ACE2 and/or transmembrane serine protease 2 (TMPRSS2) genes from humans and other animal species in the avian fibroblast cell line, DF1, that is not permissive to infection. We demonstrated that expression of both human ACE2 and TMPRSS2 genes is necessary to support SC2 infection and replication in DF1 and a non-permissive sub-lineage of MDCK cells. Titers of SC2 in these cell lines were comparable to those observed in control Vero cells. To further test the model, we developed seven additional transgenic cell lines expressing the ACE2 and TMPRSS2 derived from Felis (cat), Equus (horse), Sus (pig), Capra (goat), Mesocricetus (Golden hamster), Myotis lucifugus (Little Brown bat) and Hipposideros armiger (Great Roundleaf bat) in DF1 cells. Results demonstrate permissive replication of SC2 in cat, Golden hamster, and goat species, but not pig or horse, which correlated with the results of reported challenge studies. The development of this cell culture model allows for more efficient testing of the potential susceptibility of many different animal species for SC2 and emerging variant viruses. IMPORTANCE SARS-CoV-2 (SC2) is believed to have originated in animal species and jumped into humans where it has produced the greatest viral pandemic of our time. Identification of animal species susceptible to SC2 infection would provide information on potential zoonotic reservoirs, and transmission potential at the human-animal interface. Our work provides a model system to test the ability of the virus to replicate in an otherwise non-permissive cell line by transgenic insertion of the ACE2 and TMPRSS2 genes from human and other animal species. The results from our in vitro model positively correlate with animal infection studies enhancing the predicative capability of the model. Importantly, we demonstrate that both proteins are required for successful virus replication. These findings establish a framework to test other animal species for susceptibility to infection that may be critical zoonotic reservoirs for transmission, as well as to test variant viruses that arise over time.

The SARS-CoV-2 (SC2) virus has caused a worldwide pandemic because of the 23 virus's ability to transmit efficiently human-to-human. A key determinant of infection is the 24 attachment of the viral spike protein to the host receptor angiotensin-converting enzyme 2 25 (ACE2). Because of the presumed zoonotic origin of SC2, there is no practical way to assess 26 every species susceptibility to SC2 by direct challenge studies. In an effort to have a better 27 predictive model of animal host susceptibility to SC2, we expressed the ACE2 and/or 28 transmembrane serine protease 2 (TMPRSS2) genes from humans and other animal species in 29 the avian fibroblast cell line, DF1, that is not permissive to infection. We demonstrated that 30 expression of both human ACE2 and TMPRSS2 genes is necessary to support SC2 infection and The main surface protein of CoVs is the spike (S) protein that facilitates receptor binding 78 and fusion of the viral lipid envelope with the host cell membrane. Receptor binding is facilitated 79 by the S1 subunit while the S2 subunit is involved with fusion of the viral membrane with the 80 cell membrane (14, 15) . For these two events to occur, the S protein needs to be post-81 transitionally modified by two different host proteases to become activated. For SC2, furin-like 82 proteases cleave the S protein at the S1/S2 site that contains a multiple basic amino acid motif 83 (RRAR) that is different from SARS-CoV (16). The S protein undergoes additional cleavage at 84 the S2' site by the cellular type II transmembrane serine protease, . However, 85 other proteases have been described to activate CoVs including cathepsin L, TMPRSS11A and 86 TMPRSS11D (20-23).

SARS-CoV and SC2 utilize the angiotensin-converting enzyme 2 (ACE2) as the receptor 88 for attachment on host cells with the S protein (14). ACE-2 is a single-pass type I transmembrane 89 protein, with its enzymatically active domain exposed on the surface of cells in lungs and other 90 tissues. ACE2 catalyzes the conversion of angiotensin I into angiotensin 1-9 and angiotensin II 91 into angiotensin1-7, which are involved with vasodilation effects in the cardiovascular system 92 (24, 25). Due to conservations of the ACE2 gene among animal species, the potential host range 93 of SC2 is thought to be extensive.

The ACE2 and TMPRSS2 genes have homologues in many animal species (1, 22).

Several species, including house cats, ferrets, and golden hamsters, have been shown to be 96 naturally and/or experimentally infected with SC2 (26). These three species have >80% 97 sequence similarity in their ACE2 and TMPRSS2 genes when compared to the human genes.

The chicken, which does not appear to be a susceptible host, has an ACE2 homology of less than 99 70% to the human gene (27). However other species like pigs have a sequence similarity of 100 6 >80%, but are poorly susceptible to infection. Based on previous work with SARS-CoV, the 101 binding of S1 to ACE2 can be defined by the interaction of relatively few amino acids, and 102 predictions of host susceptibility based on these interactions have been made (1, 28). Despite the 103 clear importance of the binding of the spike protein to ACE2, the prediction of host susceptibility 104 does involve other factors including the level and tissue distribution of ACE2 expression and the 105 requirement for protease activation.

Because chickens are not susceptible to SC2 virus, and their ACE2 and TMPRSS2 107 protease are distinctly different from the human equivalents, we developed an avian cell line to 108 screen the potential host range of infection of the virus through the expression the ACE2 and 109 TMPRSS2 genes from human and animal species to provide novel insights into the receptor 110 usage, replication and potential host range of SC2 These studies were designed to determine if 111 the host restriction is strictly from the difference in the receptor and/or protease. One long-term 112 goal of this work is to develop a predictive framework for improved epidemic surveillance to 113 include protection of agriculturally relevant species and animal species that are hard to test 114 experimentally. Polybrene (8µg/ml) was added to each transduction reaction, supplied from the manufacturers, to 146 aid with membrane charge. Cells were incubated at 39°C for 72 hours after which media was 147 removed and replaced with fresh media containing 10% FBS. Transduction was confirmed using 

Primers used for human ACE2 PCR were Forward 5' CTA GCT GTC AAG CTCTTC CTG 212 GCT C 3' and Reverse 5' GGA TCC TAA AAG GAG GTC TGA ACA TCA TCA 3'. Reaction 213 conditions were 98°C for thirty seconds, followed by 35 cycles of 98° for ten seconds, 68°C for 214 thirty seconds and 72°C for one minute, after which a final extension of ten minutes at 72° was Table 2 . Figure 2 ). Detection of the inserted genes was confirmed with RT-PCR using 323 primers specific for the human and chicken genes (Figure 2A and B) . Expression of human 324 ACE2 and human TMPRSS2 protein in DF1 ++ and MDCK ++ cells was confirmed via western 325 blot ( Figure 2C ). activate SC-2. It is worth mentioning that proteolytic cleavage of the S protein at the S1/S2 412 20 interface was assumed to be provided by furin-like enzymes naturally present in the DF1 or 413 MDCK cell lines.

The SC2 utilizes the ACE2 protein as the primary receptor for entry into host cells and 415 the TMPRSS2 protease has been shown to be critical for cleavage/activation of the spike protein 416 (32, 33). In these studies, the transgenic insertion of the human ACE2 and TMPRSS2 genes 

Broad host range of 500

SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in vertebrates

The SARS-CoV-2 Spike protein has a broad tropism for mammalian 507 ACE2 proteins

A novel coronavirus associated with severe 513 acute respiratory syndrome

The human coronavirus HCoV-229E S-protein structure

TMPRSS2 and furin are both essential for proteolytic 591 activation of SARS-CoV-2 in human airway cells

TMPRSS11A activates the influenza A virus 595 hemagglutinin and the MERS coronavirus spike protein and is insensitive against 596 blockade by HAI-1

Role of the ACE2/Angiotensin

Axis of the Renin-Angiotensin System in Heart Failure

Angiotensin-converting enzyme 2 overexpression in the subfornical organ prevents 603 the angiotensin II-mediated pressor and drinking responses and is associated with 604 angiotensin II type 1 receptor downregulation

Animal Reservoirs and Hosts for Emerging 608 Alphacoronaviruses and Betacoronaviruses

Lack of Susceptibility to SARS-CoV-2 and MERS-CoV in Poultry

Acute Respiratory Syndrome Coronavirus 2

Return of the Coronavirus: 2019-nCoV

Vaccination with virus-like particles containing H5 antigens from three H5N1 623 clades protects chickens from H5N1 and H5N8 influenza viruses

US 627 CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute 628 Respiratory Syndrome Coronavirus 2

The first few days of a SARS-CoV-2 infection 631 viewed at single-cell resolution

SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in 635 vitro during propagation in TMPRSS2-deficient cells

Link of a ubiquitous human coronavirus to dromedary camels

Zoonotic origins of human 644 coronaviruses

Phenotypic 650 and genetic characterization of MERS coronaviruses from Africa to understand their 651 zoonotic potential

Infection of bat and human intestinal organoids by SARS-CoV-2

Respiratory Syndrome Coronavirus 2 from Patient with Coronavirus Disease, United 664 States

Characterization of spike 668 glycoprotein of SARS-CoV-2 on virus entry and its immune

Detection and Isolation of 674 SARS-CoV-2 in Serum, Urine, and Stool Specimens of COVID-19 Patients from the 675 Republic of Korea

SARS-CoV-2 spike protein-mediated cell signaling in lung 679 vascular cells

Identification of SARS-CoV-2 inhibitors using lung and colonic 686 organoids

Inhibition of SARS-CoV-2 691 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2

Yuen 695 KY. 2020. A broad-spectrum virus-and host-targeting peptide against respiratory viruses 696 including influenza virus and SARS-CoV-2

SARS-CoV-2 productively infects human gut 702 enterocytes

SARS-CoV-2 in fruit bats, ferrets, pigs, and chickens: an experimental 707 transmission study

Functional and genetic analysis of viral receptor ACE2 orthologs 717 reveals a broad potential host range of SARS-CoV-2

SARS CoV-2 Spike Protein in silico Interaction With ACE2 Receptors From Wild and 720 Domestic Species

Susceptibility of swine cells and domestic pigs to 725 SARS-CoV-2

Pigs are not susceptible to SARS-CoV-2 infection but are a 730 model for viral immunogenicity studies

Susceptibility of Domestic Swine to Experimental Infection with Severe Acute 734 Respiratory Syndrome Coronavirus 2

Evaluating angiotensin-converting enzyme 2-mediated 738 SARS-CoV-2 entry across species

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Binding 742 to ACE2 Receptors from Human, Pets, Farm Animals, and Putative Intermediate Hosts

and Vero cells were inoculated with SC2 at multiplicity of 769 infection (MOI) of 1. At time points indicated, supernatant samples were taken for RNA 770 extraction and determination of viral titers by RT-PCR. The values shown are mean +/-standard 771 deviation of triplicate samples. Two-way analysis of variance with Tukeys multiple comparison 772 test was performed on titers at 48 hours post inoculation to determine the statistical difference in 773 virus titer between the cell lines. Lines with different lowercase letters indicate differences 774 (p<0.05). (B) Pass 2 of virus from cell culture lines expressing human ACE2, TMPRSS2, or 775 both. After 72 hours of growth, supernatants of pass 1 were transferred onto fresh monolayers of 776 cells, allowed to absorb for 1 hour and removed. Fresh media was added and samples were taken 777 at time points indicated to determine virus titer by RT-PCR

MDCK, and MDCK expressing both human 783 ACE2 and TMPRSS2 (++) were grown at 37C in 5% CO 2 on glass chamber slides. Cells were 784 inoculated with SC2 at MOI of 1. At 48 hours post inoculation monolayers were examined for 785 cytopathic effect and detection of virus with rabbit monoclonal antibodies against SC2 spike and 786 nucleoprotein. Cells were washed 3 times with PBS and incubated in the secondary antibody, 787 goat anti-rabbit IgG H&L (Alexa Fluor® 555) for one hour at room temperature

Little Brown bat 794 (Myotis lucifugus) and Great Roundleaf bat (Hipposideros armiger). Cells were first created with 795 the animal ACE2 gene and FACS purified based on GFP expression. The animal TMPRSS2 796 gene was then transfected into the DF1 cells expressing the animal ACE2 gene. Two-color 797 FACS was performed based on GFP and RFP expression

ACE2 and TMPRSS2 were grown at 37C in 5% CO 2 . After 72 hours, RNA was extracted and 799 primers specific for the animals ACE2 and animal TMPRSS2 were used with RT-PCR to 800 confirm animal species ACE2 and TMPRSS2 expression in DF1 cells.