MERS-CoV was first identified and described in November 2012 Zaki et al., 2012) from a case of pneumonia in the Kingdom of Saudi Arabia (KSA). MERS-CoV is an enveloped virus containing a 30kb single-stranded, positive-sense RNA genome. Genomic analysis revealed that MERS-CoV is a novel member of the Coronaviridae closely related to two known bat coronaviruses (BtCoV), BtCoV-HKU4 and BtCoV-HKU5, so has been placed with these viruses in lineage C of the Betacoronavirus genus .
The development of assays for growth and quantification of MERS-CoV has been helped by previous work on other human and animal coronaviruses. In the wake of the outbreak of severe acute respiratory syndrome (SARS)-CoV in 2003, a number of quantitative methods were developed for SARS-CoV and these have been used as a starting point for development of assays for MERS-CoV. Chan et al., 2013) , making it possible to develop assays for MERS-CoV in vitro. MERS-CoV does not replicate in small animals (Coleman et al., 2013; de Wit et al., 2013a) and only in rhesus macaques (de Wit et al., 2013b) and marmosets (Falzarano et al., 2014) which are much more difficult and expensive to handle, so the ability to test the efficacy of these assays on samples from in vivo MERS-CoV infection is limited. Here we describe methods for growing (Basic Protocol 1) and quantifying (Basic Protocols 2-4) MERS-CoV in vitro.
CAUTION: MERS-CoV is a Biosafety Level 3 (BSL-3) pathogen. Follow all appropriate guidelines for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
MERS-CoV productively infects a number of cell lines; new stocks of MERS-CoV can be created by simple infection and collection of supernatant. However, it is important to note that different strains of MERS-CoV may grow at different rates, which affects the point at which supernatant should be harvested for maximum yield. MERS-CoV causes damage to and, ultimately, death of infected cells, therefore a good guide to the success of the virus growth is observation of cell death, termed the cytopathic effect (CPE).
Vero E6 cells Vero E6 cell growth media: minimal essential media (MEM) supplemented with 10% heat inactivated fetal calf serum (FCS), 1% L-glutamine and 1% penicillin/streptomycin T175 tissue culture flask 1.5ml screw cap tubes 1. Using basic cell culture methods, maintain Vero E6 cells in continuous culture for at least a week after removal from long-term storage before use and trypsinize and resuspend in normal Vero E6 cell growth media.
Seed approximately 1×10 7 Vero E6 cells into a T175 flask so that they will be 90-95% confluent the following day.
Culture overnight at 37°c in 5% CO 2 .
Remove media so that 5ml remains.
Add 1ml MERS-CoV to the flask.
Distribute virus over cells and incubate for 1 hour at 37°c in 5% CO 2 .
Replenish media up to a total volume of 20ml.
Incubate flask for 48-72 hours at 37°c in 5% CO, or until significant CPE is observed.
The appearance of CPE can be strain specific or dependent on the starting titer of the seed stock. We observed significant CPE with MERS-EMC at 48 hours post-infection, and significant CPE with MERS-Jordan at 72 hours post-infection. We would recommend checking the flask daily from 48 hours post-infection onwards.
Collect supernatant from the infected flask and centrifuge at 500g for 5 minutes to remove any cellular debris.
Aliquot appropriate volumes (100µl-1ml) of the clarified supernatant into 1.5ml screw cap tubes and store at −80°c until needed.
Each tube should only be used once for subsequent infections as freeze:thawing can significantly decrease the infectious titer. 1ml tubes are useful for re-infecting for the next round of growth, though bear in mind that MERS-CoV, in common with all RNA viruses, will mutate as it is passaged.
Basic Protocol 2: Quantification of MERS-CoV by tissue culture infective dose 50 (TCID 50 )
The tissue culture infective dose that causes 50% cytotoxicitiy (TCID 50 ) assay is a quantitative method for assessing the infectivity of a virus stock. 1 TCID 50 is defined as the amount of pathogen that causes death of 50% of cells (Reed, 1938) , so depends on the ability of the virus to kill the cells in culture. Here we describe a method for crystal violet staining of a TCID 50 , however there are other vital cell dyes or methods for determining cell viability that can be used instead.
Vero E6 Resuspend 1×10 6 cells in 10ml per 96-well plate.
Dilute sample(s) in normal Vero E6 growth media and then continue diluting down a 5-or 10-fold dilution series, ideally in duplicate or triplicate.
For ease, samples can be diluted in a duplicate 96-well plate without cells. Change tips between each dilution.
3. Transfer 100µl of the diluted MERS-CoV sample to each well to the cells in the 96well plate.
The same tip(s) can be used when going up the dilution series.
CPE can be strain specific. We observed the best TCID 50 results at 48 hours post-infection for MERS-EMC and 96 hours for MERS-Jordan.
Remove media from cells and fix cells in 100µl ice-cold 4% paraformaldehyde (PFA) for 5 minutes at room temperature.
Remove PFA and stain cells with 100µl 0.05% w/v crystal violet stain in 20% methanol for 30 minutes at room temperature.
BSL-3 regulations mean that this is best done by adding 100µl of tap water to each well and then removing into an appropriate disinfecting solution.
8. Tap plate dry on tissue.
Visualize plate and score wells. Each well should be scored as positive for MERS-CoV (i.e. MERS-CoV has killed all of the cells, so there is no crystal violet stain) or negative for MERS-CoV (i.e. the cells have all survived, so the well is stained violet)
The crystal violet stain should be visible to the naked eye, but a light box is useful to discriminate wells that have light staining.
Calculate TCID 50 using the following formula: There are TCID 50 calculators available online.
Plaque assays are used to quantify a wide-range of viruses, depending on the ability of a virus to cause cell lysis and spread to neighboring cells, both of which MERS-CoV is able to do. The basic premise of a plaque assay is that the virus infects a single cell and then spreads out in a radial pattern from that initial infection if the media covering the cells is semi-solid so as not to allow free diffusion of the virus in the media. The area of cell death caused by this spread is named a plaque and each plaque can be assumed to have developed from a single cell infection by a single virion, hence the titer from this assay is in plaque forming units (pfu) per ml. Seed 1 plate per sample.
Reagents to prepare prior to starting work:
• 2 × MEM supplemented with 20% heat inactivated fetal calf serum (FCS), 2% L-glutamine, 2% penicillin/streptomycin plus 1.5ml of phenol red.
Media that contains phenol red is usually at '1x' concentration, so will be diluted into the agar, therefore we prefer to purchase phenol red free media and supplement with 2x phenol red.
• 1.6% w/v agarose in distilled water. Microwave the agarose solution for 1-2 minutes to melt the agarose and place in a 65°c water bath to maintain as a liquid 2. Dilute samples in PBS down a 10-fold dilution series, giving enough to add 200µl per remaining volume.
3. Add 200µl of each dilution to one well of a 6-well plate.
Incubate plate at 37°c in 5% CO 2 for 1hour, shaking every 15 minutes.
Mix the 2× supplemented MEM with the 1.6% agarose in a 1:1 ratio.
6. Add 3ml of MEM:agarose mix into each well of the 6-well plates.
Work quickly but smoothly to ensure the agarose mix does not set prior to addition 7. Incubate plates at 37°c in 5% CO 2 for 3 days.
Optional: add 3ml neutral red stain to each well and incubate for 1-2 hours 8. Visualize and count plaques. Count the plaques at the dilution at which there are 10-100 plaques.
For those used to counting plaques of SARS-Co, MERS-CoV are smaller and more difficult to make out.
Calculate titer in pfu/ml using the following formula:
Tier (pfu/ml) = number of plaques × 1/ dilution factor × 1/volume (ml)
Coronaviruses contain a single-stranded positive-sense RNA genome which is transcribed into negative-sense RNA intermediates for replication and protein production. For basic coronavirus detection, primers can be designed to part of the genomic RNA, which will detect both the genomic RNA and the corresponding mRNA for that open reading frame (ORF).
Coronavirus mRNA is made as a sub-genomic positive-sense RNA that contains a common 5' primer leader sequence derived from the 5' end of the genomic RNA, followed by the ORF of the viral gene (reviewed in Pasternak et al., 2006) . Therefore, it is possible to discriminate coronavirus mRNA from genomic RNA by PCR by designing the following primers: 1) a forward primer containing part of the leader sequence and 2) a reverse primer in a gene that is not immediately 3' to the leader sequence so that the genomic RNA is not detected (i.e. anything other than the sequence for the ORF1a replicase polyprotein).
MERS-CoV is currently not classified as a Select Agent, which means that the RNA containing solutions from MERS-CoV infected cells can be handled under BSL-2 conditions as long as it is confirmed that all live MERS-CoV have been destroyed. In this assay, we have designed TaqMan® primer/probe sets to an area upstream of the MERS-CoV envelope gene (UpE), to the membrane protein (M) mRNA are shown in Table 1 . An appropriately labeled endogenous control can also be added to the mix to make a triplex assay, for example we have used an ABY-labeled human transferrin receptor protein 1 (TFRC) primer/ probe set with success on human and monkey samples.
RNA sample. Cell culture extract or homogenized tissue in Trizol® or similar.
Optional/required: reagents for cDNA synthesis. For example, RevertAid reverse transcriptase (Thermo Scientific) MERS-CoV primer/probe sets described in Table 1 TaqMan® mastermix reagent We use fast 1-step mix (Life Technologies), to which RNA can be added directly and does not require a separate cDNA synthesis step.
Run on PCR machine capable of reading FAM, VIC and ABY. Run parameters of temperature and cycle time may vary with different Taqman® reagents, so instructions provided with reagent should be followed.
Calculate ΔΔCt using the following calculation:
Step 1: ΔCt:
Step 2: ΔΔCt
Step 3: transformation to relative expression
Background Information-MERS-CoV is an emerging human beta-coronaviruses that was first detected in KSA in November 2012 . As of December 2014, there have been 904 confirmed MERS-CoV cases, with 347 deaths. MERS-CoV infections have been detected in 23 countries, primarily in the Middle East, but also in Europe, South-East Asia and North America (www.coronamap.com).
Members of the Coronaviridae virus family are enveloped viruses with a large singlestranded positive sense RNA genome. The coronaviruses genome encodes structural proteins: membrane (M), spike (S), envelope (E) and nucleocapsid (N); two replicase polyproteins: ORF1a and ORF1b and between one and eight accessory proteins that perform important functions in coronavirus replication and pathogenesis in vivo, such as blocking innate immune signaling pathways (Frieman and Baric, 2008) . In the case of MERS-CoV, there are 5 accessory proteins , for which mechanisms of action are starting to be characterized (Matthews et al., 2014; Niemeyer et al., 2013; Siu et al., 2014; Yang et al., 2013) .
MERS-CoV is not the first highly pathogenic coronavirus to emerge, or indeed the first pathogenic coronavirus. In 2002, highly pathogenic SARS-CoV emerged in China and spread around the world before disappearing again in 2003 and there are also less pathogenic human coronaviruses, such as hCoV-NL-63 and hCoV-OC43. Therefore, the coronaviruses have become an important group of viruses for scientific research. Given the background of coronavirus research and that MERS-CoV replicates in a wide-range of cell types in vitro, assays for MERS-CoV growth and quantification have been rapidly developed.
Tissue culture problems: MERS-CoV relies on healthy cells in order to propagate, so any issues with cell culture can dramatically affect the MERS-CoV yield. Bacterial and fungal contamination of cell culture media can be avoided by adding antibiotics (for example penicillin and streptomycin) and/or anti-fungals to the media. Good asceptic tissue culture technique, such as wearing gloves and appropriate PPE, spraying with 70% ethanol and not waving hands over uncapped tubes or tissue culture bottles should reduce contamination. Stored cell culture media should be regularly inspected for signs of contamination (cloudiness or fungal outgrowth) and disposed of if found to be contaminated.
Vero E6 cells do not overgrow plates as readily as other cell types, as they can slow down cell division once they become confluent. However, it remains possible to overgrow them and kill them, so maintain vigilance of the cells in culture and if they are over-confluent prior to infection, re-seed a fresh flask/plate of cells.
The MERS-CoV RNA detection assay described in Basic Protocol 4 is very sensitive and we have been able to detect MERS-CoV RNA in cells that are less susceptible to MERS-CoV.
Time Considerations-For all protocols (Basic Protocols 1, 2 and 3) involving the handling of live MERS-CoV must be completed under BSL-3 conditions. Preparing to enter a BSL-3 environment can take 10-20 minutes and careful preparation is required to collect together any reagents, equipment and cells required to be taken into the BSL-3 laboratory. Under current regulations, Basic Protocol 4 can be performed under BSL-2 conditions once the Trizol® has been harvested from cells, however if MERS-CoV becomes a Select Agent, then MERS-CoV RNA will have to be handled under BSL-3 or Select Agent BSL-2 conditions, which will add time to Basic Protocol 4.
For Basic Protocols 1, 2 and 3, the longest time will be spent waiting for CPE in the infected cells -this can take 3-4 days for MERS-CoV, depending on strain. However, for MERS-CoV replication assays, we have detected infectious, though low titer, MERS-CoV in the supernatant from infected cells as early as 24 hours post-infection. For Basic Protocol 4, we have been able to detect MERS-CoV products of replication as early as 12 hours postinfection in MERS-CoV infected cells.
The infection of cells for Basic Protocol 1 should take no longer 90 minutes, with the majority of the time taken up with the 1-hour initial infection. Harvesting of virus in Protocol 1 includes aspiration of media, centrifugation and aliquoting into appropriate tubes, which takes around 60 minutes total. The dilutions required for Basic Protocols 2 and 3 can take anywhere from 10 minutes to 1 hour, depending on the number of samples, a multichannel pipette significantly shortens the time that would otherwise be required to do each sample individually. Basic Protocol 3 requires a 1-hour incubation with MERS-CoV prior to adding the agarose gel solution to the plates. The crystal violet stain for Basic Protocol 2 takes 1-2 hours, depending on the number of plates, including the 30 minutes required for the stain itself. Reading of plaques for Basic Protocol 3 takes approximately 10-30 minutes, depending on the number of plates.
RNA isolations for Basic Protocol 4 can take anywhere from 10 minutes to 2.5 hours depending on the number of samples and the RNA isolation method used. If a cDNA is required prior to PCR in Basic Protocol 4, this can take approximately 2 hours, depending on number of samples and reagents used, though the resulting cDNA can be left at 4°c at least overnight, so the reaction can be set up late in the day and left overnight with an infinite 4°c step as the last step. Similarly, preparing and running the PCR plate for Basic Protocol 4 can take anywhere from 1.5 hours to 3 hours, depending on the number of samples and the Taqman® reagent used.