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BioMed Central Virology Journal Open Access Methodology Plaque assay for human coronavirus NL63 using human colon carcinoma cells Petra Herzog 1 3 Christian Drosten 2 and Marcel A M ller 2 Address 1 Bernhard Nocht Institute for Tropical Medicine Bernhard Nocht Str 74 D 20359 Hamburg Germany 2 Institute of Virology University of Bonn Medical Centre Sigmund Freud Str 25 53127 Bonn Germany and 3 Qiagen Hamburg GmbH K nigstr 4a D 22767 Hamburg Germany Email Petra Herzog herzog bni hamburg de Christian Drosten drosten virology bonn de Marcel A M ller muller virology bonn de Corresponding author Abstract Background Coronaviruses cause a broad range of diseases in animals and humans Human coronavirus hCoV NL63 is associated with up to 10 of common colds Viral plaque assays enable the characterization of virus infectivity and allow for purifying virus stock solutions They are essential for drug screening Hitherto used cell cultures for hCoV NL63 show low levels of virus replication and weak and diffuse cytopathogenic effects It has not yet been possible to establish practicable plaque assays for this important human pathogen Results 12 different cell cultures were tested for susceptibility to hCoV NL63 infection Human colon carcinoma cells CaCo 2 replicated virus more than 100 fold more efficiently than commonly used African green monkey kidney cells LLC MK2 CaCo 2 cells showed cytopathogenic effects 4 days post infection Avicel agarose and carboxymethyl cellulose overlays proved suitable for plaque assays Best results were achieved with Avicel which produced large and clear plaques from the 4 th day of infection The utility of plaque assays with agrose overlay was demonstrated for purifying virus thereby increasing viral infectivity by 1 log 10 PFU mL Conclusion CaCo 2 cells support hCoV NL63 better than LLC MK2 cells and enable cytopathogenic plaque assays Avicel overlay is favourable for plaque quantification and agarose overlay is preferred for plaque purification HCoV NL63 virus stock of increased infectivity will be beneficial in antiviral screening animal modelling of disease and other experimental tasks Background Coronaviruses are large enveloped plus strand RNA viruses that are currently classified in three groups or pre sumptive genera 1 3 Group 1 is further divided into two phylogenetic clades exemplified by the transmissible gas troenteritis virus TGEV and the porcine epidemic diar rhoea virus PEDV respectively The latter clade contains two prototypic human coronaviruses hCoV termed genic 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 iden tified hCoV HKU1 and NL63 are major causes of com mon colds in wintertime 10 HCoV NL63 was isolated Published 12 November 2008 Virology Journal 2008 5 138 doi 10 1186 1743 422X 5 138 Received 22 October 2008 Accepted 12 November 2008 This article is available from 2008 Herzog et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License http creativecommons org licenses by 2 0 which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited Page 1 of 9 page number not for citation purposes hCoV 229E and NL63 4 5 Like group 1 group 2 con tains mammalian CoV These include two human patho in African green monkey kidney cells LLC MK2 from a seven month old infant with bronchiolitis and conjuncti Virology Journal 2008 5 138 vitis 4 In further investigations the virus was predomi nantly 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 corona viral 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 immunos taining 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 purifi cation 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 parti cles 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 con tributes 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 test ing different overlays and assay formats we developed cytopathogenic NL63 plaque assays that can be used for analytical and preparative purposes Results and discussion Susceptibility of different cell lines to hCoV NL63 and cytopathogenic effects LLC MK2 and Vero cells do not cause clear CPE on infec tion with hCoV NL63 Because this virus uses the same receptor as the SARS CoV 12 different cell cultures sus ceptible to SARS CoV infection were tested for susceptibil ity to hCoV NL63 34 36 37 Table 1 Cells in six well plates were infected with 10e4 plaque forming units of Table 1 Comparison of hCoV NL63 replication by real time RT PCR using different cell cultures Designation Day 0 copies L Day 7 copies L Amplification factor Cytopathogenic effect CPE Vero E6 6 94e3 3 05e7 4 39e3 None Vero FM 1 78e4 4 51e9 2 54e5 None CaCo 2 3 55e3 1 25e10 3 54e6 round and detached dead cells with cell debris in supernatant strong effect Calu1 2 61e4 5 33e6 2 04e2 None Calu6 7 95e3 5 00e5 6 29e1 None POEK 8 11e4 3 03e5 3 74e0 None PK13 2 66e2 7 78e5 2 93e3 None 293lp 3 67e3 3 09e7 8 42e3 None FeA 1 45e4 5 83e5 4 03e1 None RD 3 14e5 1 57e4 4 99e 2 None PS 1 20e4 1 44e6 1 19e2 None LLC MK2 4 00e3 2 65e6 6 62e2 round and detached weak effect Vero E6 rhesus kidney cells ATCC CRL 1586 Vero FM rhesus kidney cells ATCC CCL 81 CaCo 2 human colon carcinoma ATCC HTB 37 Calu 1 human lung carcinoma ICLC HTL95002 Calu 6 human lung carcinoma ICLC HTL97003 POEK porcine foetal kidney cell culture collection of the Robert Koch Institute RKI Berlin Germany PK13 porcine kidney cell culture collection of the Bernhard Nocht Institute BNI Hamburg Germany 293 human embryonic kidney ATCC CRL 1573 FEA feline embryonic fibroblast kindly provided by Dr Marcel Asper Page 2 of 9 page number not for citation purposes NewLab Inc Cologne RD human rhabdomyosarcoma cells RKI PS porcine kidney cells RKI and LLC MK2 African green monkey kidney cells ATTC CCL 7 kindly provided by Lia van der Hoek Academic Medical Center Amsterdam The Netherlands Virology Journal 2008 5 138 hCoV NL63 virus stock LLC MK2 NP RNA concentra tions in supernatants were measured short after virus adsorption i e in fresh medium after washing off of the infection supernatant and 7 days later Table 1 Increase of virus RNA was less than 1000 fold in seven of 12 cul tures Interestingly this included LLC MK2 the prototype cell culture for NL63 In spite of a low amplification factor these cells showed the usual weak CPE that is typically observed when infected with hCoV NL63 Vero cells seemed to support virus growth efficiently but produced no CPE Interestingly there was a remarkable difference between Vero E6 and Vero FM cells Table 1 In our hands these cells also showed differences in growth of SARS CoV Vero FM consistently showed more pro nounced CPE than Vero E6 but there were no significant differences in RNA amplification not shown CaCo 2 cells amplified virus RNA most efficiently and showed a clearly visible CPE starting from day 4 after infection Cells became rounded detached from the sur face and showed morphological signs of cell death Fig ure 1 For confirmation of differential replication efficiencies CaCo 2 and LLC MK2 cells were infected in parallel Both cell lines were seeded in 25 cm 2 flasks and infected at multiplicities of infection of 0 005 Samples of superna tants were taken daily from day 0 to 7 and analyzed by real time RT PCR As shown in Figure 2 CaCo 2 cells repli cated virus more efficiently than LLC MK2 From day 3 onward RNA concentrations were more than 100 fold higher in CaCo 2 cells Because of the clear CPE observed in CaCo 2 cells these cells were tested for their utility in a cytopathogenic plaque assay Comparison of different overlays Three overlay techniques commonly used for plaque assays were tested for their suitability 23 CaCo 2 cells were infected in 6 well plates with hCoV NL63 After one hour supernatants were removed cells washed with PBS and overlaid as follows For CMC overlays 1 mL fresh DMEM was added to each well Subsequently 1 mL of 1 6 CMC solution was slowly added per well Agarose overlays 1 final concen tration were prepared by melting 2 agarose at 70 C cooling it in a water bath to 42 C and mixing it immedi ately before application with an equal volume of 2 DMEM stored at room temperature Two mL of the mix ture were carefully applied to each well Avicel overlays were made by mixing 2 4 Avicel solution with an equal volume of 2 DMEM 2 mL of the mixture were immedi ately added to each well Plaque assays were incubated without disturbing at 37 C and 5 CO 2 Overlays were removed on day five and cells were fixed with a solution of 4 formaldehyde in PBS After 30 min the formaldehyde solution was removed cells were washed twice with PBS and stained with a 0 2 crystal violet solution As shown in Figure 3 plaques were Cytopathogenic effect of hCoV NL63 on human colon carcinoma cells CaCo 2 Figure 1 Cytopathogenic effect of hCoV NL63 on human colon carcinoma cells CaCo 2 CaCo 2 cells 5 days after infec tion with hCoV NL63 at an multiplicity of infection of 0 1 agarose overlay technique A mock infection B infection Photo Page 3 of 9 page number not for citation purposes graphs were taken at 40 fold magnification bars represent 20 m Virology Journal 2008 5 138 visible with all three overlays but staining was clearest with Avicel Incubation times HCoV NL63 culture with LLC MK2 cells takes more than 7 days until first signs of weak CPE become visible In order to test whether incubation times could be reduced with CaCo 2 cells five plaque assays on virus dilution series were done with Avicel overlays and terminated by fixation after 1 2 3 4 and 5 days respectively On days 1 and 2 no plaques were visible not shown Termina tion at day 3 yielded plaques only at high virus concentra tion Figure 4 From day 4 onward plaques were visible in the lowest detectable virus concentration Plaques on day 5 were larger but did not increase in number Plaque preparation infectious virus solutions our standard virus stock LLC MK2 NP see Materials and Methods section was plaque purified using the agarose overlay Because life staining of cells with neutral red solution was not successful on CaCo 2 cells not shown we used an alternative tech nique of plaque preparation Limiting dilution infections were done on 6 well plates After 5 days cytopathic foci were identified by scanning through the wells with an inverted microscope at low magnification lighting through the clear agarose overlay The positions of CPE foci were marked with a permanent marker it was helpful to turn up the microscope light for this The agarose overlay was penetrated with a pipette and 10 to 20 l of fluid was aspirated underneath the overlay This fluid was resuspended in 100 l of Opti Pro serum free medium which served as the starting solution Growth kinetics of hCoV NL63 on LLC MK2 and CaCo 2 cellsFigure 2 Growth kinetics of hCoV NL63 on LLC MK2 and CaCo 2 cells 25 cm 2 flasks of LLC MK2 or CaCo 2 cells were infected at multiplicities of infection of 0 005 for 1 h washed twice with PBS and subsequently supplied with 10 mL DMEM Samples were taken daily from day 0 to 7 except day 4 and analyzed by real time RT PCR Error bars indicate ranges of three independent experiments Page 4 of 9 page number not for citation purposes Work with hCoV NL63 is complicated by low infectious titers in virus stock solutions In order to obtain more for a new limiting dilution infection series in the next 6 well plate plaque assay Three rounds of purification were Virology Journal 2008 5 138 done After the last round aspirated fluid was inoculated in 5 mL of Opti Pro serum free medium which was then overlaid on confluent CaCo 2 cells in a 25 cm 2 flask for infection After infection for one hour and washing 5 mL DMEM were added and flasks were incubated at 37 C 5 CO 2 for four days Stocks were harvested and stored as described for the original LLC MK2 stock in the Materials and Methods section The purified virus is hereafter To compare the infectivity of the plaque purified virus with the original LLC MK2 virus stock see Materials and Methods section viral titres were determined by Avicel plaque assay as shown in Figure 5 CaCo 2 PP was about 10 fold more infectious than LLC MK2 NP Plaque assays were repeated three times not shown Mean titres were determined to be 1 4 10e6 PFU mL and 1 3 10e5 PFU mL respectively for CaCo 2 PP and LLC MK2 NP Abso lute quantification of virus RNA by real time RT PCR yielded 4 8 10e11 RNA copies mL for CaCo 2 PP and 5 3 10e10 copies mL for LLC MK2 NP It was interesting to note that both virus stocks had rather high RNA concentrations as opposed to their infectivities PFU RNA ratios were 2 92 10e 6 for CaCo 2 PP and 2 45 10e 6 for LLC MK2 NP This high excess of RNA over infectious units might be attributable to the virus harvesting procedure possibly releasing nonpackaged RNA along with virus particles during freeze thawing Because PFU RNA ratios were very similar for both stocks it appeared unlikely that elimination of defective interfer ing particles had contributed the gain of infectivity It will be interesting in future studies to see whether hCoV NL63 might have adapted to CaCo 2 cells during plaque purifi cation Conclusion CaCo 2 cells seem to support hCoV NL63 replication sig nificantly 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 favoura ble for plaque quantification whereas agarose overlays are preferred for plaque purification Virus stock of increased infectivity will be beneficial for antiviral screen ing animal modelling of disease and other experimental tasks Methods Cell cultures All cells were cultivated in DMEM Dulbecco s Modified Eagles Medium PAA C lbe Germany with 4 5 g L Glu cose PAA supplemented with 10 Foetal Bovine Serum GOLD PAA 1 Penicillin Streptomycin 100 con centrate Penicillin 10000 U mL Streptomycin 10 mg mL PAA 1 L Glutamine 200 mM 1 Sodium Pyru vate 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 Plaque assay for hCoV NL63 on CaCo 2 cells using different overlaysFigure 3 Plaque assay for hCoV NL63 on CaCo 2 cells using different overlays HCoV NL63 was serially diluted on CaCo 2 cells 10e 1 until 10e 5 After 1 h of virus adsorb tion different overlays were added After 5 days cells were fixed with 4 formaldehyde and stained with 0 2 crystal violet solution A carboxymethyl cellulose B agarose C Avicel Page 5 of 9 page number not for citation purposes referred to as CaCo 2 PP for plaque purified mechanical re suspension Virology Journal 2008 5 138 Page 6 of 9 page number not for citation purposes Plaque assays with different incubation timesFigure 4 Plaque assays with different incubation times Plaque assays were performed with Avicel overlay and incubated for 3 4 and 5 days respectively The dilution factor of LLC MK2 NP virus stock used for infection is shown on the bottom Virology Journal 2008 5 138 Page 7 of 9 page number not for citation purposes Effect of plaque purificationFigure 5 Effect of plaque purification A plaque assay with Avicel overlay on purified virus stock CaCo 2 PP B plaque assay on non purified virus stock LLC MK2 NP C viral RNA copies per mL of supernatant left and plaque forming units per mL of super natant right for CaCo 2 PP and LLC MK2 NP virus stocks log scale Error bars show ranges of three independent experi ments Virology Journal 2008 5 138 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 recov ering it three times from the last well of a dilution series still showing diffuse CPE Subconfluent LLC MK2 monol ayers were infected in 75 cm 2 flasks with virus supernatant from the last round of limiting dilution culture at a ratio of 1 100 200 l virus supernatant in 20 mL of fresh medium This concentration was the highest virus dilu tion still infectious in this culture format The flasks were incubated at 37 C 5 CO 2 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 L GIBCO Opti Pro serum free medium Invitrogen Karl sruhe Germany plus 1 Penicillin Streptomycin PAA and 1 L Glutamine PAA was added to each well Inoc ulum 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 abso lute virus RNA quantification was performed as described previously 38 Media and overlays for plaque assays A 2 4 w v suspension of Avicel RC 581 FCM BioPol ymer Brussels Belgium was prepared in distilled wate