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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

Highlights • Delayed brain tumor (DBT) mouse cell lines stably expressing the murine angiotensin converting enzyme 2 (mACE2) have been generated.• The cell lines are highly susceptible to mouse-adapted SARS-CoV infection.• SARS-CoV-MA15 efficiently induced the expression of proinflammatory cytokines and IFN-β in DBT-mACE2 cells.• DBT-mACE2 cells provide a good experimental system that is species-homologous to the in vivo systems for evaluating SARS-CoV-host interaction studies.


Introduction
Severe acute respiratory syndrome coronavirus (SARS-CoV) is an enveloped plus-strand RNA virus of the Coronaviridae family, genus ␤, within the Nidovirales order (de Groot et al., 2012;Enjuanes et al., 2008). SARS-CoV was first detected in late 2002 in Guangdong province, China and spread to more than 30 countries in a few months, causing 8000 infections and 800 deaths Fouchier et al., 2003;Ksiazek et al., 2003;Kuiken et al., 2003;Marra et al., 2003;Peiris et al., 2003;Rota et al., 2003). The spread of the virus was ultimately controlled by the isolation of infected individuals, and the WHO declared the end of the SARS epidemic in July 2003. However, SARS-like coronaviruses remain and are circulating in bats all over the world, making virus reemergence a realistic possibility (Lau et al., 2005;Li et al., 2005;Woo et al., 2006).
Coronaviruses encode two overlapping open reading frames (ORFs 1a and 1b) that are translated into two polyproteins which are processed by two viral proteases to yield 16 non-structural replicase proteins (Ziebuhr et al., 2000). These proteins are involved in genome replication and transcription of subgenomic mRNAs encoding the structural proteins nucleocapsid (N), envelope (E), membrane (M) and spike (S), as well as a set of CoV speciesspecific proteins. The spike protein is localized at the surface of the virion, and is responsible for the attachment to the cellular receptor, and for virus-cell membrane fusion, to facilitate virus entry (Gallagher and Buchmeier, 2001). The cellular receptor for SARS-CoV is the angiontesin convertin enzyme 2 (ACE2) Wong et al., 2004), although the glycoprotein CD209L (L-SIGN) may also be used as a weaker alternative receptor (Jeffers et al., 2004).
SARS-CoV infects many experimental animals such as mice, ferrets, cats, hamsters and non-human primates (cynomolgus and rhesus macaques, African green monkeys and marmosets) Subbarao and Roberts, 2006). However, none of the infection models completely reproduce human clinical disease and pathological findings. To overcome these limitations, SARS-CoV was adapted to grow in mice by passing the virus in lung for 10, 15, or 25 times (Day et al., 2009;Nagata et al., 2008;Roberts et al., 2007). Infection of Balb/c mice with the resulting mouse adapted (MA) viruses reproduced many aspects of human SARS, including pathological changes in the lung, viremia, neutrophilia, and lethality (Day et al., 2009;Nagata et al., 2008;Roberts et al., 2007). This inbred mouse model of human SARS disease has many advantages compared to the other animal models, such as small animal size, low cost, availability of the animals, the possibility to genetically manipulate the host animals (i.e. to develop gene knock-outs and knock-ins), and the availability of immunological and molecular biology reagents specific to the host animals.
Several established cell lines from different species, including monkey cells Vero E6, MA104 and FRhK-4, human cells Caco-2, CL-14, LoVo and Huh-7, pig cells PK-15, POEK and PS, and mink cells Mv 1 Lu (Chan et al., 2004;Cinatl et al., 2004;Hattermann et al., 2005;Mossel et al., 2005;Ng et al., 2003) are susceptible to SARS-CoV infection. However, not all of these cell lines possess the full complement of genes encoding innate immune signaling and response factors, and as such, some of the cell lines are not suitable for evaluating the innate host response to SARS-CoV. Mouse DBT cells have been transfected with human or civet SARS-CoV receptor ACE2 (Becker et al., 2008;Sheahan et al., 2008a,b). However, as far as we know, the susceptibility of these cell lines to a mouse adapted SARS-CoV has not been determined. In addition, mouse cell lines have never been stably transfected with the mouse ACE2, which is the natural receptor used by the mouse adapted SARS-CoV.
In this work, we established mouse cell lines stably expressing the murine SARS-CoV receptor ACE2 Wong et al., 2004). These cell lines were highly susceptible to mouse adapted SARS-CoV infection and were also transfectable with high efficiency. Using these cell lines, we demonstrated that a mouse adapted SARS-CoV (SARS-CoV-MA15) induced the expression of genes leading to inflammation, as shown in SARS patients and experimental animal models infected with SARS-CoV. In addition, SARS-CoV-MA15 induced a weak IFN-␤ response, as generally shown in coronavirus infections, preventing the robust production of type I IFNs. These mouse cell lines are valuable tools to perform in vitro studies that could be further developed in the species-homologous SARS mouse models.

Cells
Delayed brain tumor (DBT) cells were originally obtained from an intracerebral tumor induced in an adult BALB/c mouse by intracerebral injection of Rous sarcoma virus (Kumanishi, 1967). The African green monkey kidney-derived Vero E6 cells were kindly provided by Snijder (University of Leiden, The Netherlands). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 25 mM HEPES, 10% fetal bovine serum (FBS, Biowhittaker), and 1% non-essential amino acids (SIGMA) and incubated in a 5% CO 2 atmosphere, at 37 • C.

Plasmids
The plasmid pcDNA3.1 encoding the murine ACE2 gene (GeneBank sequence NM 001130513.1) fused to 5 myc tag codons (pcDNA3.1-myc-mACE2), and a gene providing geneticin (G418) resistance in prokaryotic and eukaryotic cells, was kindly provided by Farzan (Harvard Medical School, USA) . SARS-CoV full-length cDNAs encoding the Urbani strain or the mouse adapted strain (MA15) were assembled in bacterial artificial chromosomes (BACs) under the control of the cytomegalovirus (CMV) immediate-early promoter to allow the expression of the viral RNA in the nucleus by cellular RNA polymerase II. At the 3 end, these cDNAs were flanked by a 25-bp poly(A) tail, followed by the hepatitis delta virus ribozyme and the bovine growth hormone and termination and polyadenylation sequences, to generate a correct 3 end (Almazan et al., 2006(Almazan et al., , 2000.  were rescued from infectious cDNA clones generated (Almazan et al., 2006;Fett et al., 2013). To recover infectious viruses, BHK cells were grown to 95% confluence in a 25-cm 2 flask and transfected with the cDNA clones by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's specifications. At 6 h posttransfection (hpt), cells were trypsinized, plated over a confluent monolayer of Vero E6 cells grown in a 25-cm 2 flask, and incubated at 37 • C for 72 h. After one passage in Vero E6 cells, the recovered viruses were cloned by three rounds of plaque purification. All work with infectious viruses was performed in biosafety level (BSL) 3 plus facilities. All personnel were equipped with positive-pressure air purifying respirators (3 M HEPA AirMate, St. Paul, MN).

Generation of DBT cells stably expressing mACE2
To transfect DBT-mACE2 with high efficiency, 3 × 10 5 DBT-mACE2 cells were transfected using the program EN-158 of Amaxa 4D-Nucleofector and 1 g of the linearized plasmid pcDNA3.1myc-mACE2 in buffer SG (Lonza). Nucleofected cells were seeded on 24-well plates. After nucleofection, cells were incubated for 10 min at room temperature (RT), and then the cells were added to DMEM (GIBCO) supplemented with 25 mM HEPES, 10% fetal bovine serum (FBS, Biowhittaker) and 1% non-essential amino acids (SIGMA) and incubated at 37 • C. 24 h post-nucleofection, G418 was added to the culture medium, to a final concentration of 800 g/ml, to select for geneticin resistance. The selective medium was changed every 3-4 days for 2 weeks, and the cells were cloned three times by limiting dilution (1 cell/well) in the presence of G418 (800 g/ml). Subsequently, ten clones of DBT-mACE2 were amplified in the presence of 800 g/ml of G418.

Transfection of DBT-mACE2 clones
To analyze the efficiency of transfection, DBT-mACE2 cells were nucleofected as described above, using a GFP-encoding plasmid. 24 h post nucleofection GFP expression was analyzed by fluorescence activated cell sorting (FACS). DBT-mACE2 cells were transfected with an efficiency higher than 90%.

Indirect immunofluorescence microscopy
To detect viral N proteins and the myc tag fused to mACE2, DBT-mACE2 cells were grown to 80% confluence on glass coverslips and infected with rSARS-CoV-MA15 at a moi of 0.1. At 24 hpi, media were removed and cells were washed twice with PBS and fixed and permeabilized with ice-cold 100% methanol for 20 min at −20 • C or with 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 min at room temperature, in the case of non-permeabilized cells. Primary antibody incubations were performed in PBS containing 10% FBS for 90 min at room temperature. The mouse mAb SA46-4 specific for N protein, kindly provided by Fang (South Dakota State University, Brookings, USA) (dilution 1:500), and a mouse anti-myc (dilution 1:500, Millipore, Ref. 05-724), were used. Coverslips were washed two times with PBS between primary and secondary antibody incubations. Secondary antibodies (Invitrogen) were Alexa 488 (for detection of N protein) or Alexa 594 (for detection of myc tag) conjugates and were incubated for 45 min at room temperature at 1:500 dilutions in PBS containing 10% FBS. Nuclei were stained using DAPI (dilution 1:200, Sigma). Coverslips were mounted in Prolong Gold anti-fade reagent (Invitrogen) and examined on a Leica SP5 confocal microscope (Leica Microsystems). The percentage of mACE2 and viral N-positive cells were calculated by analyzing 10 random fields, each one containing at least 30 cells.

Virus production in different DBT-mACE2 clones
DBT-mACE2 clones grown to densities of 3.0 × 10 5 cells/cm 2 were infected at a moi of 0.1 with SARS-CoV-MA15 or SARS-CoV-Urbani. Culture supernatants were collected at 72 hpi, and virus titers were determined in Vero E6 cells as previously described (DeDiego et al., 2007).
To analyze the effect of the moi on virus production, DBT-mACE2 clone 6 cells, which supported the highest viral titers, were infected at mois ranging from 0.0001 to 1 pfu per cell with the rSARS-CoV-MA15 virus. Culture supernatants were collected at the indicated times post-infection and titrated on Vero E6 cells.
To determine the effect of cell density on virus production, DBT-mACE2 clone 6 cells at different cell densities were infected at a moi of 0.1. Culture supernatants were collected at 72 hpi and titrated in Vero E6 cells.

Virus titration in DBT-mACE2 cells
DBT-mACE2 clone 6 cells at a cell density of 2.5 × 10 5 cells/cm 2 were infected with SARS-CoV-MA15. Cell cultures were incubated at 37 • C for 60 min for virus adsorption and then overlaid with DMEM containing 0.6% low melting agarose and 4% FBS. 48 hpi, cells were fixed with 10% formaldehyde in PBS and stained with a solution containing 0.1% (w/v) crystal violet and 20% methanol.

Expression of inflammation-related cytokines and IFN-ˇ and -in rSARS-CoV-MA15 infected cells
DBT-mACE2 clone 6 cells were infected with rSARS-CoV at a moi of 0.1. Total RNAs from DBT-mACE2 infected cells were extracted at 48 hpi using the Qiagen RNeasy kit according to the manufacturer's instructions. Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) reactions were performed at 37 • C for 2 h with a High Capacity cDNA transcription kit (Applied Biosystems) using 100 ng of total RNA and random hexamer oligonucleotides. Cellular gene expressions were analyzed using TaqMan gene expression assays (Applied Biosystems) specific for Mus musculus genes (Table 1). Data were acquired with an ABI PRISM 7000 sequence detection system (Applied Biosystems) and analyzed with ABI PRISM 7000 SDS version 1.0 software. Gene expression in mock-infected cells and rSARS-CoV-MA15infected cells was compared. Quantification was achieved using the 2 − Ct method, which analyzes relative changes in gene expression in qPCR experiments (Livak and Schmittgen, 2001). The results of three independent experiments were analyzed. DBT-mACE2 clone 6 cells were infected at a moi of 0.1 and cell extracts in lysis buffer (1% NP-40, 50 mM Tris-HCl, pH 7.6, 2 mM NaCl, 2 mM EDTA and protease inhibitors) were prepared at 48 hpi. Cell extracts were diluted 1:5 in assay buffer (Millipore). The expression of mouse CXCL10/IP-10 and CXCL2/macrophage inflammatory protein 2 (MIP-2) was evaluated with the Luminex technology and a mouse cytokine antibody bead kit (Milliplex map kit; Millipore), following the manufacturer's instructions. Data were collected from three independent infections.

Generation of mouse DBT cells expressing the SARS-CoV receptor mACE2
To generate a mouse cell line susceptible to SARS-CoV infection, DBT cells were nucleofected with a plasmid encoding the SARS-CoV receptor mACE2 fused N-terminally to a myc tag (myc-mACE2) along with an antibiotic resistance marker. Transfected cells were selected by using geneticin (G418). Ten independently-derived cell clones were generated. mACE2 expression in the different clones was analyzed by immunofluorescence using an antibody specific for the myc tag. The ten selected clones showed high mACE2 expression in more than 70% of the cells (Fig. 1A and B). The myc-mACE2 expression was stable for at least 20 passages in tissue culture (data not shown). Myc-mACE2 was detected in the plasma membrane even in non-permeabilized cells, confirming that mACE2 is present at the cell surface. As expected, myc-mACE2 was undetectable in untransfected DBT cells (Fig. 1A and B). To determine whether the DBT-mACE2 clones were easily transfected with plasmid DNAs (a useful method for future studies), the cells were nucleofected with a plasmid encoding green fluorescent protein (GFP). Interestingly, the efficiency of DBT-mACE2 nucleofection was greater than 90% (data not shown).

Susceptibility of DBT-mACE2 to SARS-CoV infection
To determine whether the DBT-mACE2 cells were susceptible to SARS-CoV, the 10 selected clones, untransfected DBT cells, and positive-control Vero E6 cells were infected with the mouse adapted SARS-CoV-MA15 strain at a moi of 0.1. No infectious virus was recovered from untransfected DBT cells at 72 hpi ( Fig. 2A). Interestingly, in transfected cells, all DBT-mACE2 clones showed high viral titers at 72 hpi, ranging from 5.0 × 10 6 to 1.2 × 10 7 pfu/ml, respectively. DBT-mACE2 clones 1 and 6 produced the highest virus Table 1 Taqman assays used to analyze the expression of cellular genes by quantitative RT-PCR. outputs (9.2 × 10 6 and 1.2 × 10 7 pfu/ml), which were as high as those obtained in Vero E6 cells ( Fig. 2A).
To analyze whether DBT-mACE2 cells were also susceptible to a non-mouse adapted SARS-CoV, the clone 6 of DBT-mACE2 cells, producing the highest SARS-CoV-MA15 titers, untransfected DBT cells and Vero E6 cells were infected with SARS-CoV-Urbani. Infectious viruses were also recovered in DBT-mACE2 cells, although with titers more than 10 3 -fold lower than those for SARS-CoV-MA15 (Fig. 2B). In contrast, no infectious virus was recovered from untransfected DBT cells. SARS-CoV-Urbani and MA15 virus titers in Vero E6 cells were similar for both viruses ( Fig. 2A and B).
To identify conditions for optimal virus production, moi and cell density parameters were varied. Firstly, DBT-mACE2 clone 6 cells were infected with SARS-CoV-MA15 at mois ranging from 0.0001 to 1. Virus titers were determined at 0, 4, 24, 48 and 72 hpi (Fig. 3). The highest viral titer (>10 7 pfu/ml) was obtained at moi of 0.1. Virus  titers in cells infected with a moi of 1 rapidly decreased after 24 hpi, probably due to extensive cell death. Indeed, considerable cytopathic effect (CPE) was observed by microscopy. DBT-mACE2 clone 6 cells were also infected at cells densities ranging from 0.5 × 10 5 and 4 × 10 5 cells/cm 2 , at the optimal moi of 0.1, and virus titers were determined at 72 hpi. The highest viral titers (>10 7 pfu/ml) were observed at a cell density of 3 × 10 5 cells/cm 2 (Fig. 4).
To determine the percentage of infected cells, untransfected DBT cells, and DBT-mACE2 clones were infected with SARS-CoV-MA15, and analyzed by immunofluorescence with an antibody specific for the viral N protein ( Fig. 1A and B). The percentage of infected cells, detected by the presence of N protein, was higher than 70% in all the DBT-mACE2 transfected clones.
SARS-CoV infection caused CPE in DBT-mACE2 cells. At input mois of 0.1, cell rounding and detachment was evident at 48 hpi, being almost total at 72 hpi. Control uninfected cells showed no morphological changes (Fig. 5A). In fact, SARS-CoV-MA15 formed  clear, easily identifiable plaques in DBT-mACE2 cell monolayers. Lysis plaques on DBT-mACE2 cells and Vero E6 cells were compared. Smaller plaques were observed using Vero E6 cells in comparison to DBT-mACE2 cells (Fig. 5B). Virus titers obtained by plaque assays on Vero E6 and DBT-mACE2 indicator cells were very similar (4.3 × 10 7 and 4.0 × 10 7 , respectively).
To evaluate whether the induction of cytokine mRNAs in SARS-CoV-MA15-infected cells correlate with an induction at the protein level, the levels of CXCL10 and CXCL2 proteins were determined in mock and DBT-mACE2-infected cells. In agreement with the results obtained with the mRNAs, CXCL10 and CXCL2 were also upregulated at the protein level, showing fold-increases of 3 and 1.8-fold over mock, respectively (Fig. 6B). These data indicated that DBT-mACE2 cells were useful to study proinflammatory cytokine expression in vitro.

Discussion
The development of established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection is described in this study. The best current small animal model for SARS-CoV is the infection of Balb/c mice with a mouse adapted SARS-CoV (Day et al., 2009;Nagata et al., 2008;Roberts et al., 2007). The establishment of mouse cell lines susceptible to SARS-CoV was therefore of high interest, so that in vitro and in vivo evaluations with the mouse adapted SARS-CoV could take place in the same species environment and using the same cellular receptor.
The expression of mACE2 was sufficient to convert DBT cells, which were not susceptible to SARS-CoV infection, into cells producing high SARS-CoV-MA15 titers. Similarly, the transient expression of mACE2 in mouse 3T3 cells generated susceptibility to SARS-CoV infection, although these cells were not stably transformed . In addition, the expression of civet and human ACE2 in DBT cells (Becker et al., 2008;Sheahan et al., 2008a,b), and the expression of human ACE2 in mice also led to cells susceptible to SARS-CoV and to an animal model more susceptible to the infection by a human SARS-CoV (McCray et al., 2007;Yang et al., 2007). All these data indicated that ACE2 is clearly a required SARS-CoV receptor.
SARS-CoV-Urbani titers in DBT-mACE2 cells were more than 10 3 -fold lower than titers obtained for SARS-CoV-MA15. Critical mutations differentiating these two virus strains map in the gene encoding the spike protein  that binds to the cellular ACE2 and facilitates virus entry . The affinity of SARS-CoV-Urbani spike protein binding to mACE2 is lower that the affinity of SARS-CoV-MA15 spike protein binding to mACE2, making the Urbani virus entrance less efficient .
SARS-CoV infection in DBT-mACE2 cells induced high levels of CPE, probably due to apoptosis, as it has been observed in other SARS-CoV-infected cell lines, such as Vero (DeDiego et al., 2011;Ren et al., 2005;Yan et al., 2004). This virus-induced cytopathology provides an additional experimental advantage of DBT-mACE2 cell lines, as the infection outcomes are overt and are similar to what happens to the epithelial respiratory cells in SARS patients (Lang et al., 2003;Zhang et al., 2003). Accordingly, plaque assays of SARS-CoV-MA15 were efficient in DBT-ACE2 cells.
The DBT-mACE2 cells generated in this work, could be used in the context of virus adaptation to a homologous receptor of another host species, such as for example hamsters or rats and in the context of mouse adaptation to the murine receptor. In addition, DBT-mACE2 cells may be useful to grow mouse adapted SARS-CoV, with reduced likelihood of undesired changes in the SARS-CoV genome that may occur during replication in non-murine cells.