A rapid and affordable in vitro model to test SARS-CoV-2 susceptibility in animal species

By Dr. Chinta SidharthanReviewed by Benedette Cuffari, M.Sc.Apr 24 2023

In a recent study posted to the bioRxiv* preprint server, researchers developed a novel in vitro model that expresses the angiotensin-converting enzyme 2 (ACE-2) receptor from seven bar species in a non-permissive avian fibroblast cell line to study the susceptibility to and replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 

Study: SARS-CoV-2 utilization of ACE2 from different bat species allows for virus entry and replication in vitro. Image Credit: Olena.Hypnosis / Shutterstock.com

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

The etiological agent of the coronavirus disease 2019 (COVID-19), SARS-CoV-2, belongs to the Coronaviridae family and is an enveloped, single-stranded ribonucleic acid (RNA) virus.

Other coronaviruses, such as the Middle East respiratory syndrome coronavirus (MERS-CoV), have previously caused epidemics with high fatalities among humans. Both MERS-CoV and SARS-CoV are believed to have spread from bats to humans.

The natural host of SARS-CoV-2 is also hypothesized to be bats. However, while SARS-CoV-2 has been found circulating among Rhinolophus bats, the progenitor virus of SARS-CoV-2 has yet to be identified.

Among the 1,400 species of bats found around the world, the susceptibility to SARS-CoV-2 has only been tested in four species. While testing the vulnerability of each of these bat species to SARS-CoV-2 might not be feasible, in vitro models that express ACE-2 receptors can be used to test the ability of the virus to replicate in specific bat species.

About the study

In the present study, an in vitro model consisting of a non-permissive chicken cell line (DF1) was used to express ACE-2 receptors from seven bat species to test their susceptibility to SARS-CoV-2.

ACE-2 genes were obtained from GenBank for seven bat species, including Myotis lucifugus (little brown bat), Hipposideros armiger (great roundleaf bat), Rhinolophus pearsonii (Pearson's horseshoe bat), Rhinolophus ferrumequinum (greater horseshoe bat), Tadarida brasiliensis (Brazilian free-tailed bat), Rousettus aegyptiacus (Egyptian rousette), and Rhinolophus sinicus (Chinese rufous horseshoe bat).

Fluorescence activation cell sorting (FACS) was used to identify transgenic DF1 cells expressing human transmembrane serine protease 2 (TMPRSS2) and bat ACE-2 receptor. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to determine the expression levels of bat ACE-2 in transgenic cells, while immunofluorescence and Western blot assays were used to measure the expression of TMPRSS2 and ACE-2.

DF1 cells expressing ACE-2 receptors from each of the seven bat species were infected with SARS-CoV-2. Whereas qRT-PCR was used to detect viral replication, microscopy was used to evaluate the cytopathic effects of infection.

Importantly, TMPRSS2 and ACE-2 protein sequences from bats and humans were aligned and compared for similarity.

Study findings

The expression of ACE-2 receptors from seven bat species in DF1 cells caused the cells to be susceptible to three SARS-CoV-2 isolates. Notably, viral replication levels differed between bat species and the tested SARS-CoV-2 variants.

The Wuhan-Hu-1 strain of SARS-CoV-2 exhibited the highest replication titers. When human ACE-2 receptors were expressed in DF1 cells instead of bat ACE-2, the replication titers of all viral variants were high.

The seven bat species included in this study vary considerably in their foraging habits, roosting sites, and distribution ranges, which can impact the likelihood of their contact with humans.

Although little brown bats, Brazilian free-tailed bats, and Egyptian rousettes are believed to have more contact with humans, horseshoe bats are found in more rural areas with less contact with humans. The ACE-2 receptor from the Chinese rufous horseshoe bat, Pearson's horseshoe bat, and greater horseshoe bat allowed the entry and replication of all three SARS-CoV-2 variants.

The researchers also infected Egyptian rousettes with the Wuhan strain of SARS-CoV-2 and observed viral titers in their respiratory tracts, as well as the presence of neutralizing antibodies against SARS-CoV-2. Similarly, the ACE-2 receptor from Egyptian rousettes expressed in DF1 cells supported high replication titers of the SARS-CoV-2 Wuhan strain, whereas the titers for the Delta and Lambda variants were lower.

Conclusions

The novel in vitro model described in the current study was effective in testing these species' susceptibility to SARS-CoV-2.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.