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

Abstract

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.

Susceptibility of Domestic Goat (Capra aegagrus hircus) to Experimental Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) B.1.351/Beta Variant

“…Similarity between goat and human ACE2 receptors has been demonstrated via comparative genomic analysis as well as binding affinity between goat ACE2 and SARS-CoV-2 S protein [42]. Other previous in vitro studies also reported that goat ACE2 supports cell entry of SARS-CoV-2 [43,44], and its replication has been demonstrated in DF1 cells expressing both goat ACE2 and transmembrane serine protease 2 (TMPRSS2) [43]. Since the susceptibility of animal species could change after the appearance of SARS-CoV-2 variants and our in silico study predicted potential affinity between the B.1.351/Beta variant and ACE2 goat receptor, the aim of the present study was to investigate the susceptibility of goat to this variant of SARS-CoV-2 by means of an in vivo experimental inoculation.…”

SARS-CoV-2 utilization of ACE2 from different bat species allows for virus entry and replicationin vitro

“…Analysis of bat ACE2 genes. We obtained ACE2 protein sequences from seven different bat species for phylogenetic analysis and compared them to other animals previously tested (34).…”

“…The copyright holder for this preprint (which this version posted April 20, 2023. ; https://doi.org/10.1101/2023.04. 19.537521 doi: bioRxiv preprint universal bat ACE2 primers, and chicken 28S primers were used as previously described (34).…”

“…For each cell line, media was removed from three wells and 0.4 mL of virus inoculum was added. Virus preparation was performed as previously reported (34). The same volume of sterile medium was used as a sham inoculated control.…”

“…19.537521 doi: bioRxiv preprint for SC2 susceptibility (31)(32)(33). In this work, we expanded on previous studies looking at species susceptibility to SC2 using a chicken cell line that is nonpermissive to SC2 replication (34).…”