key: cord-0865595-maiif36b
authors: Kapczynski, Darrell R.; Sweeney, Ryan; Spackman, Erica; Pantin-Jackwood, Mary; Suarez, David L.
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: 2022-02-12
journal: Virology
DOI: 10.1016/j.virol.2022.01.014
sha: 92e8b04f73ccb212e8b6d661de20e3e5abb18d05
doc_id: 865595
cord_uid: maiif36b

The SARS-CoV-2 (SARS-CoV-2) 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 SARS-CoV-2, there is no practical way to assess the susceptibility of every species to SARS-CoV-2 by direct challenge studies. In an effort to have a better predictive model of animal host susceptibility to SARS-CoV-2, 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 SARS-CoV-2 infection and replication in DF1 and a non-permissive sub-lineage of MDCK cells. Titers of SARS-CoV-2 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 catus (cat), Equus caballus (horse), Sus domesticus (pig), Capra hircus (goat), Mesocricetus auratus (Golden hamster), Myotis lucifugus (Little Brown bat) and Hipposideros armiger (Great Roundleaf bat) in DF1 cells. Results demonstrate permissive replication of SARS-CoV-2 in cat, Golden hamster, and goat species, but not pig or horse, which correlated with the results of reported challenge studies. Cells expressing genes from either bat species tested demonstrated temporal replication of SARS-CoV-2 that peaked early and was not sustained. The development of this cell culture model allows for more efficient testing of the potential susceptibility of many different animal species for SARS-CoV-2 and emerging variant viruses.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which was first reported in Wuhan, China in late 2019. This virus 48 most probably has its ecological reservoir in bats, and transmission of the virus to humans has actin (Invitrogen). The blot was washed as before, incubated for 1 hour, at room temperature, in 214 secondary antibody diluted 1:20,000 in TBS with gentle rocking. Secondary antibodies included 215 rat anti-mouse IgG1 HRP (Southern Biotech, Birmingham, AL), and mouse anti-rabbit IgG1 216 HRP (Southern Biotech). After incubation, the blot was washed 3 times as above in TBST.

Pierce ECL substrate (Fisher) was added to the blot for 1 minute and excess was removed by 218 gentle wicking. The blot was placed into an x-ray cassette and exposed to x-ray film (Fisher) for 219 1 minute, developed and fixed (Kodak).

For immunohistochemistry of SARS-CoV-2 replication, cells were seeded into an I-Bidi 221 8-well chambered slide (Fisher) at a density of 4 X 10 4 in 500 ul DMEM containing 10% FBS 222 and grown overnight as above. When cells reached 75% confluence the media was removed, and virus was added at MOI of 1 as above. After 48 hours, the media was removed and cells were 224 fixed for 5 minutes at 4C in 1:1 ice cold ethanol:methanol. Cells were then washed twice with 225 cold PBS as above. Cells were blocked as above for one hour at room temperature then washed 3 growth (Han et al., 2021; Harcourt et al., 2020; Kim et al., 2020; Lamers et al., 2020; Monteil et 362 al., 2020; Ou et al., 2020; Suzuki et al., 2021; Zhao et al., 2020; Zhou et al., 2020) . However, 363 because these systems can naturally be infected, they are not useful for testing host susceptibility 

TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal 702 enterocytes

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Binding 706 to ACE2 Receptors from Human, Pets, Farm Animals, and Putative Intermediate Hosts

Evaluating angiotensin-converting enzyme 2-711 mediated SARS-CoV-2 entry across species

A broad-spectrum virus-and host-targeting peptide against respiratory viruses 715 including influenza virus and SARS-CoV-2

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

Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat 728 origin

Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their 735 zoonotic potential

At time points indicated, supernatant samples were taken for 766 RNA extraction and determination of viral titers by RT-PCR. The values shown are mean +/-767 standard deviation of triplicate samples. Two-way analysis of variance with Tukeys multiple 768 comparison test was performed on titers at 48 hours post inoculation to determine the statistical 769 difference in virus titer between the cell lines. Lines with different lowercase letters indicate 770 differences

After 72 hours of growth, supernatants of pass 1 were transferred onto fresh 772 monolayers of cells, allowed to absorb for 1 hour and removed. Fresh media was added and 773 samples were taken at time points indicated to determine virus titer by RT-PCR. Statistical 774 analysis was performed at 48 hours post inoculation