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. 2013 Nov;193(2):639-46.
doi: 10.1016/j.jviromet.2013.07.039. Epub 2013 Aug 1.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines

Jose A Regla-Nava  1 , Jose M Jimenez-Guardeño, Jose L Nieto-Torres, Thomas M Gallagher, Luis Enjuanes, Marta L DeDiego
Affiliations

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines

Jose A Regla-Nava et al. J Virol Methods. 2013 Nov.

Abstract

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis.

Keywords: Coronavirus; Mouse adapted; Proinflammatory cytokines; SARS; SARS-CoV receptor ACE2; Stably transformed murine cells.

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Figures

Fig. 1
Fig. 1
Expression of myc-mACE2 and viral nucleoprotein in DBT-mACE2-infected clones. (A) Untransformed DBT and DBT-mACE2 cells were infected at a moi of 0.1 with rSARS-CoV-MA15. Myc-mACE2 and viral nucleoprotein expression was analyzed by indirect immunofluorescence using specific myc and nucleoprotein antibodies followed by A549 and A488-conjugated secondary mouse antibodies. Asterisks indicate cells that do not express myc-mACE2 or viral nucleoprotein, as controls. (a) myc-mACE2 in permeabilized cells, (b) viral nucleoprotein in permeabilized cells, (c) myc-mACE2 in non-permeabilized cells and (d) myc-mACE2 and nucleoprotein in untransformed DBT cells. (B) The percentage of myc-mACE2 positive and viral N protein-positive cells was calculated by analyzing 10 random fields, each one containing 30 cells.
Fig. 2
Fig. 2
SARS-CoV production in DBT-mACE2 cell clones. Cells were infected at a moi of 0.1 with rSARS-CoV-MA15 (A) or rSARS-CoV-Urbani. (B) Viral titers in cell supernatants at 72 hpi were measured using a plaque assay on Vero E6 cells. Error bars represent standard deviations of the mean from three experiments.
Fig. 3
Fig. 3
Effect of moi on the growth kinetics of SARS-CoV-MA15 in DBT-mACE2 clone 6 cells. DBT-mACE2 clone 6 cells were infected at the indicated input mois. Viral titers in cell supernatants at the indicated times post-infection were measured by plaque assay on Vero E6 cells. Error bars represent standard deviations of the mean from three experiments.
Fig. 4
Fig. 4
Effect of cell density on the growth of SARS-CoV-MA15 in DBT-mACE2 clone 6 cells. DBT-mACE2 clone 6 cells were seeded at different cell densities and infected at a moi of 0.1. Viral titers in cell supernatants at 72 hpi were measured by plaque assay on Vero E6 cells. Error bars represent standard deviations of the mean from three experiments.
Fig. 5
Fig. 5
Cytopathic effect and lysis plaques produced by SARS-CoV-MA15 on DBT-mACE2 clone 6 cells. (A) DBT-mACE2 clone 6 cells were mock-infected or infected with SARS-CoV-MA15 at a moi of 0.1. CPE was visualized at 24, 48, and 72 hpi. (B) Lysis plaques produced after 2 days by SARS-CoV-MA15 infection in DBT-mACE2 and Vero E6 cells.
Fig. 6
Fig. 6
Expression of proinflammatory cytokines in SARS-CoV-MA15-infected cells. DBT-mACE2 clone 6 cells were infected at a moi of 0.1 with rSARS-CoV-MA15. (A) Cellular RNAs were extracted at 48 hpi. The expression of the indicated cytokines and interferons, and that of 18S rRNA as a control, was determined by qRT-PCR. In each case, the corresponding mRNA expression levels in SARS-CoV-MA15-infected cells were plotted as fold-change relative to expression levels in uninfected cells. (B) Cell extracts were prepared at 48 hpi. Expression of the cytokines CXCL10 and CXCL2 at the protein level was evaluated in mock and DBT-mACE2-infected cells.
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