Surface–Aerosol Stability and Pathogenicity of Diverse Middle East Respiratory Syndrome Coronavirus Strains, 2012–2018

Neeltje van Doremalen; Michael Letko; Robert J. Fischer; Trenton Bushmaker; Jonathan Schulz; Claude K. Yinda; Stephanie N. Seifert; Nam Joong Kim; Maged G. Hemida; Ghazi Kayali; Wan Beom Park; Ranawaka A.P.M. Perera; Azaibi Tamin; Natalie J. Thornburg; Suxiang Tong; Krista Queen; Maria D. van Kerkhove; Young Ki Choi; Myoung-don Oh; Abdullah M. Assiri; Malik Peiris; Susan I. Gerber; Vincent J. Munster


Emerging Infectious Diseases. 2021;27(12):3052-3062. 

In This Article



Animal experiment approval was obtained by the Institutional Animal Care and Use Committee at Rocky Mountain Laboratories, National Institutes of Health (Hamilton, MT, USA). All animal experiments were executed in an Association for Assessment and Accreditation of Laboratory Animal Care–approved facility, following the guidelines in National Institutes of Health Guide for the Care and Use of Laboratory Animals, Animal Welfare Act, US Department of Agriculture, and United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. The Institutional Biosafety Committee approved work with MERS-CoV strains under Biosafety Level 3 conditions.

Viral Stock Propagation

We provide strain-specific details for the viruses used in this study (Table). Viruses were isolated by others and provided for this study. SARS-CoV-2/Washington was isolated by the Centers for Disease Control and Prevention (Atlanta, GA, USA).

We obtained MERS-CoV strains from the following sources: EMC12 from Erasmus Medical Center (Rotterdam, the Netherlands); U/14, KSA/15, and KSA/18 from the Centers for Disease Control and Prevention; SK/15 from Chungbuk National University (Cheongju, South Korea); and C/KSA/13, C/E/13, and C/BF/15 from Hong Kong University (Hong Kong, China). We passaged MERS-CoV and SARS-CoV-2 strains once in Vero E6 cells in Dulbecco's modified Eagle medium (DMEM; Sigma Aldrich, supplemented with 2% fetal bovine serum (Thermo Fisher Scientific,, 50 U/mL of penicillin (Thermo Fisher), and 50 μg/m of streptomycin (Thermo Fisher).

We maintained Vero E6 cells in DMEM supplemented with 10% fetal bovine serum, 1 mmol/L of l-glutamine, 50 U/mL of penicillin, and 50 μg/mL of streptomycin. We clarified virus stocks by centrifugation and froze them at −80°C. We performed virus titrations by using endpoint titration in Vero E6 cells inoculated with 10-fold serial dilutions of virus. We scored cytopathic effect at day 5 (for MERS-CoV) or day 6 (for SARS-CoV-2) and calculated median tissue culture infectious dose (TCID50) from 4 replicates by using the Spearman–Karber method.[22]

Sequencing Stocks

We treated MERS-CoV samples with RiboZero H/M/R rRNA Depletion Mix (Illumina, according to the manufacturer's instructions. After purification with Ampure RNACleanXP (Beckman Coulter, ttps://, we eluted enriched RNA and assessed it on a BioAnalyzer RNA Pico Chip (Agilent Technologies, We prepared second-strand cDNA according to the Truseq Stranded mRNA Library Preparation Guide (Illumina). We treated samples with RiboShredder RNase Blend (

We visualized final libraries on a BioAnalyzer DNA1000 Chip (Agilent Technologies), and quantified them by using a KAPA Library Quant Kit (Illumina) and a universal qPCR Mix (Kapa Biosystems, on a CFX96 Real-Time System (Bio-Rad Laboratories, We pooled libraries together in equimolar concentrations and sequenced them using MiSeq (Illumina) with on-board cluster generation and 2 × 250 paired-end sequencing. The cluster density was at 454 k/mm2/lane, resulting in 8.7 million reads passing filter/run and an average 85% greater than the Q30 score.


We downloaded all available MERS-CoV genome sequences from GenBank and curated them to remove sequences that were not independently sampled. We aligned sequences with the consensus sequences for MERS-CoV isolates used in this study by using MAFFT version 7.388 plugin[23] in Geneious Prime ( We inferred a phylogenetic tree by using the maximum-likelihood method under the general time reversible plus gamma model of nucleotide substitution with 1,000 bootstrap replicates implemented with PhyML version 3.3.20190321 (

Stability of MERS-CoV on Surfaces and in Aerosols

We sterilized 15-mm polypropylene discs (ePlastics, https://www.eplastics), AISI 304 alloy stainless steel discs (Metal Remnants,, copper discs (99.9%; Metal Remnants), and silver discs (99.9%) (Sigma-Aldrich,, placed them in 24-well plates, and added 50 μL of MERS-CoV (105 TCID50/mL). For timepoints taken at 0, 1, 24, 48, and 72 h, we added 1 mL of DMEM to wells, aliquoted, and stored at −80°C. We titrated samples on Vero E6 cells and maintained the temperature (21°C–22°C) and humidity (45%–55%).

We determined virus stability in aerosols as described.[24] In brief, we loaded a collison nebulizer with 106.5 TCID50/mL of MERS-CoV in DMEM containing 2% fetal bovine serum. Aerosols were maintained in a Goldberg drum and samples collected at 0, 30, 60, 120, and 180 min after aerosolization by passing air at a volume of 6 L/min for 30 s from the drum through a 47-mm gelatin filter (Sartorius, Filters were dissolved in 10 mL of DMEM containing 10% fetal bovine serum and stored at −80°C. All samples were titrated on Vero E6 cells.

Replication of MERS-CoV Strains in Vitro

We inoculated Vero E6 cells with virus (multiplicity of infection = 0.01) and collected supernatants at 8, 24, 48 and 72 hours postinfection (hpi). Human airway epithelium (HAE) inserts (Epithelix, were maintained as specified by the manufacturer. We washed HAEs with 200 μL of phosphate-buffered saline for 30 min, followed by inoculation with MERS-CoV at a multiplicity of infection of 0.1. We obtained samples at 8, 24, 48, 72, and 96 hpi.

Animal Experiments

We inoculated intranasally transgenic BALB/c mice expressing human DPP4 with 103 TCID50 MERS-CoV. Mice were weighed and swabbed daily. At day 3, we euthanized 4 mice and harvested lung tissue. We monitored the remaining 6 mice for survival. We euthanized mice if there were signs of severe disease signs based on quantitative assessment (e.g., hunched posture, lack of movement) or >20% weight loss.

RNA Extraction and Quantitative Reverse Transcription PCR

We homogenized lung tissues and extracted RNA by using the RNeasy method (QIAGEN, according to the manufacturer's instructions. We added swab specimens to 1 mL of DMEM, vortexed them, and used 140 μL for RNA extraction by using the QiaAmp Viral RNA Kit and a QIAxtractor (QIAGEN).

We detected MERS-CoV viral RNA by using the UpE MERS-CoV assay[25] and the Rotor-GeneTM Probe Kit (QIAGEN). Primers in this assay target a highly conserved region upstream of MERS-CoV envelope gene. Sequences of MERS-CoV strains used in this study are identical in this region. MERS-CoV dilutions with known genome copies were run in parallel to enable calculation of genome copies in samples.

Histologic and Immunohistochemical Analysis

We fixed harvested tissues for ≥7 days in 10% neutral-buffered formalin, processed them by using a VIP-6 Tissue Tek Tissue Processor (Sakura Finetek,, and embedded them in Ultraffin Paraffin Polymer (Cancer Diagnostics, We stained 5-μm sections with hematoxylin and eosin and detected coronavirus immunoreactivity by using MERS-CoV nucleocapsid protein rabbit antibody (diluted 1:4,000; Sino Biological Inc,

We processed tissues for immunohistochemical analysis by using the Discovery ULTRA Automated IHC/ISH Staining Instrument (and a Discovery ChromoMap DAB Kit (both from Ventana Medical Systems, For morphometric analysis, we scanned slides by using the Aperio ScanScope AT2 (Aperio Technologies, Inc., and analyzed the entire section by using ImageScope Positive Pixel Count Algorithm version 9.1 (Aperio Technologies, Inc.). All tissue slides were evaluated by a board-certified veterinary anatomic pathologist.

Statistical Analyses

We performed analyses by using GraphPad Prism version 7.05 for Windows ( All strains were compared with EMC/12. For aerosol stability data analysis, we determined linear regression for the mean value of 3 runs/virus. We determined statistical significance in deviation from MERS-CoV/EMC12 results by using 1-way analysis of variance, followed by the Bonferroni multiple comparisons test or a 2-way unpaired Student's t-test. We used simple linear regression to evaluate slopes of decay. Survival of mice compared with mice inoculated with EMC/12 was performed by using the log-rank (Mantel-Cox) test. To calculate the amount of virus shedding per mouse in in vivo comparisons, we calculated the area under the curve for a plot of the viral load measured in oropharyngeal swab specimens.