Background
Coronaviruses are large enveloped plus-strand RNA viruses that are currently classified in three groups or presumptive genera [
1‐
3]. Group 1 is further divided into two phylogenetic clades exemplified by the transmissible gastroenteritis virus (TGEV) and the porcine epidemic diarrhoea virus (PEDV), respectively. The latter clade contains two prototypic human coronaviruses (hCoV), termed hCoV-229E and -NL63 [
4,
5]. Like group 1, group 2 contains mammalian CoV. These include two human pathogenic prototypes, termed hCoV-OC43 and -HKU1, several important animal pathogens such as the bovine CoV and the murine hepatitis virus, as well as the SARS-CoV [
6‐
8]. Group 3 contains foremostly avian CoV [
9].
HCoV-229E and OC43 as well as the more recently identified hCoV-HKU1 and – NL63 are major causes of common colds in wintertime [
10]. HCoV-NL63 was isolated in African green monkey kidney cells (LLC-MK2) from a seven month old infant with bronchiolitis and conjunctivitis [
4]. In further investigations the virus was predominantly detected in children with respiratory infections [
11‐
14]. Up to 10% of children with respiratory disease yielded hCoV-NL63 [
10,
11,
15‐
17].
Because of its relatively high prevalence hCoV-NL63 could become an important model in screening for anti-coronaviral agents [
12,
18]. Several studies have suggested, e.g., that hCoV protease inhibitors would be cross-reactive among different hCoV [
19‐
21]. Antiviral screening relies on the detection of replicating virus in cell culture. For this and other experimental tasks, plaque assays have proven to be simple in application and efficacious in representing virus viability.
Plaque assays make use of viscous overlays to cover cells immediately after infection, thus limiting virus spread and restricting virus growth to foci of cells at the sites of initial infection. If virus contributes no or low cytopathic effects to cells, these foci may be visualized by immunostaining [
22,
23]. If virus induces strong cytopathogenic effects (CPE), cells in plaques are lysed and plaques can be visualized by staining of the residual intact cells. Cytopathogenic plaque assays are compatible with high throughput screening [
24,
25] and facilitate plaque purification and cloning of virus. This in turn is helpful in obtaining virus stocks of optimized infectivity, e.g., by decreasing the amount of defective interfering (di) particles that accumulate during serial passaging of CoV [
26].
Important technical achievements have been made in studying NL63 replication, including, most recently, the development of an infectious cDNA clone [
27]. Still it is a major obstacle that hCoV-NL63 replicates slowly and at relatively low titres in all current cell cultures, such as LLC-MK2 and Vero-B4 cells [
4,
28,
29]. Because the virus contributes very weak and diffuse CPE to these cells, there is no cytopathic plaque assay available for non-recombinant virus [
28].
Although hCoV-NL63 seems to replicate in the upper and lower airways, there are many CoV that predominantly infect the enteric tract, such as TGEV, PEDV, the feline enteric CoV, and the bovine coronavirus [
30,
31]. SARS-CoV was detected in faecal swabs from SARS patients [
32]. SARS-CoV was shown to replicate in colon carcinoma cells (CaCo-2) [
33] that are routinely used for growing entero- and adeno-, and astroviruses [
34]. Interestingly, SARS-CoV and hCoV-NL63 were shown to use the same receptor for virus entry, the angiotensin converting enzyme 2 (ACE2) [
35].
We show here that CaCo-2 cells are highly susceptible for hCoV-NL63 infections and that virus propagation in these cells is much more efficient than in LLC-MK2 cells. By testing different overlays and assay formats we developed cytopathogenic NL63 plaque assays that can be used for analytical and preparative purposes.
Conclusion
CaCo-2 cells seem to support hCoV-NL63 replication significantly better than hitherto used culture cells. Their application for a cytopathogenic plaque assay facilitates quantification of infectivity and enables studies using plaque morphology. Short incubation time of 4 days is compatible with high-throughput applications such as drug screening. The use of Avicel as an overlay is favourable for plaque quantification, whereas agarose overlays are preferred for plaque purification. Virus stock of increased infectivity will be beneficial for antiviral screening, animal modelling of disease, and other experimental tasks.
Methods
Cell cultures
All cells were cultivated in DMEM (Dulbecco's Modified Eagles Medium) (PAA, Cölbe, Germany) with 4.5 g/L Glucose (PAA), supplemented with 10% Foetal Bovine Serum "GOLD" (PAA), 1% Penicillin/Streptomycin 100 × concentrate (Penicillin 10000 U/mL, Streptomycin 10 mg/mL) (PAA), 1% L-Glutamine 200 mM, 1% Sodium Pyruvate 100 mM (PAA), 1% MEM nonessential amino acids (NEAA) 100 × concentrate (PAA). Utilized cell cultures are identified in Table
1. For passaging, cells were detached using trypsin-EDTA (PAA), except CaCo-2 cells. These were routinely subcultured by scraping and pipetting for mechanical re-suspension.
HCoV-NL63 virus stock solution
An eighth passage virus stock of hCoV-NL63 was kindly provided by Lia van der Hoek, AMC Amsterdam. It was grown in LLC-MK2 cells in limiting dilution series, recovering it three times from the last well of a dilution series still showing diffuse CPE. Subconfluent LLC-MK2 monolayers were infected in 75 cm2 flasks with virus supernatant from the last round of limiting dilution culture at a ratio of 1:100 (200 μl virus supernatant in 20 mL of fresh medium). This concentration was the highest virus dilution still infectious in this culture format. The flasks were incubated at 37°C, 5% CO2, and harvested on day four. For harvesting, flasks were frozen at -70°C and thawed. Cells and supernatant were centrifuged for 10 min at 5000 rpm. Cleared supernatant was aliquoted and stored at -70°C. This virus stock is hereafter referred to as LLC-MK2 NP (for non-purified).
Infection of cells
Cells were seeded in 6-well plates at approximately 4 × 10e5 cells per well and incubated until the monolayer was 70–80% confluent. CaCo-2 cells were grown to 100% confluence. Prior to infection cells were washed with 1 × phosphate buffered saline (PBS). Virus inoculum in 900 μL GIBCO Opti Pro serum free medium (Invitrogen, Karlsruhe, Germany) plus 1% Penicillin/Streptomycin (PAA) and 1% L-Glutamine (PAA) was added to each well. Inoculum was removed after one hour of incubation. Cells were washed twice with 1 × PBS and supplemented with 2 mL DMEM per well.
RNA extraction and real time RT-PCR
Viral RNA was extracted from cell culture supernatant with the QIAamp Viral RNA mini Kit (QIAGEN, Hilden, Germany). Real time RT-PCR for hCoV-NL63 with absolute virus RNA quantification was performed as described previously [
38].
A 2.4% (w/v) suspension of Avicel RC-581 (FCM BioPolymer, Brussels, Belgium) was prepared in distilled water and autoclaved (20 min 121°C)[
23]. A 2% (w/v) suspension of agarose (Plaque Agarose, Biozym, Hessisch Oldendorf, Germany) was prepared in distilled water and autoclaved. A 1.6% carboxymethyl cellulose (CMC) solution was prepared by autoclaving CMC powder (BDH, Poole, UK) with a magnetic stirrer. Autoclaved powder was hydrated in DMEM at 1.6% (w/v) and stirred overnight until homogenous.
Double concentrated Dulbecco's modified Eagle medium (DMEM) was prepared by mixing DMEM (PAA) with 9.48 g/L DMEM Powder (Biochrom, Berlin, Germany), supplemented with 20% Foetal Bovine Serum "GOLD" (PAA), 2% Penicillin/Streptomycin 100 × concentrate (Penicillin 10000 Units/mL, Streptomycin 10 mg/mL) (PAA), 2% L-Glutamine 200 mM, 2% Sodium Pyruvate 100 mM (PAA), 2% MEM NEAA 100 × concentrate (PAA). Medium was sterilized by filtration.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
PH performed the experiments and wrote the manuscript. CD coordinated the experiments and wrote the manuscript. MAM performed the experiments and wrote the manuscript.