key: cord-262045-r2iqpmmc authors: Smits, Saskia L.; Raj, V. Stalin; Pas, Suzan D.; Reusken, Chantal B.E.M.; Mohran, Khaled; Farag, Elmoubasher A.B.A.; Al-Romaihi, Hamad E.; AlHajri, Mohd M.; Haagmans, Bart L.; Koopmans, Marion P. title: Reliable typing of MERS-CoV variants with a small genome fragment date: 2014-12-15 journal: J Clin Virol DOI: 10.1016/j.jcv.2014.12.006 sha: doc_id: 262045 cord_uid: r2iqpmmc BACKGROUND: Middle East Respiratory Syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes lower respiratory tract infection in humans. Camels are the likely animal source for zoonotic infection, although exact transmission modes remain to be determined. Human-to-human transmission occurs sporadically. The wide geographic distribution of MERS-CoV among dromedary camels and ongoing transmissions to humans provides concern for the evolution of a MERS-CoV variant with efficient human-to-human transmission capabilities. Phylogenetic analysis of MERS-CoV has occurred by analysis of full-length genomes or multiple concatenated genome fragments, which is time-consuming, costly and limited to high viral load samples. OBJECTIVE: To develop a simple, reliable MERS-CoV variant typing assay to facilitate monitoring of MERS-CoV diversity in animals and humans. STUDY DESIGN: Phylogenetic analysis of presently known full-length MERS-CoV genomes was performed to identify genomic regions with sufficient phylogenetic content to allow reliable MERS-CoV variant typing. RT-PCR assays targeting these regions were designed and optimized. RESULTS: A reverse-transcription PCR assay for MERS-CoV targeting a 615 bp spike fragment provides a phylogenetic clustering of MERS-CoV variants comparable to that of full-length genomes. The detection limit corresponds to a cycle treshold value of ∼35 with standard upE real time PCR assays on RNA isolated from MERS-CoV EMC. Nasal swabs from RT-PCR positive camels (Ct values 12.9–32.2) yielded reliable sequence information in 14 samples. CONCLUSIONS: We developed a simple, reliable MERS-CoV variant typing assay which is crucial in monitoring MERS-CoV circulation in real time with relatively little investment on location. Middle East Respiratory Syndrome coronavirus (MERS-CoV; Family Coronaviridae) may cause severe lower respiratory tract infection in humans [1, 2] . Camels are considered the likely animal source for zoonotic infection; sporadic human-to-human transmission does occur, but is considered to be inefficient based on currently available data [3] [4] [5] [6] [7] [8] [9] [10] . Until recently, new MERS-CoV infections in humans were reported at a steady low rate reaching ∼200 confirmed human cases early 2014. In March and April 2014, however, a surge of MERS-CoV infections occurred mainly in hospitals around Jeddah, Kingdom of Saudi Arabia (KSA) and United Arab Emirates (UAE). This increased case load was in part attributed to an increase in community cases, but mostly to transmission within hospitals, with no evidence for evolution of a MERS-CoV variant with more efficient human-to-human transmission capabilities (WHO;http://www.who.int/csr/disease/coronavirus infections/ MERS CoV Update 09 May 2014.pdf?ua=1). The ongoing occurrence of new cases, however, and the finding that MERS-CoV is endemic in dromedary camels in a wide geographic region [3, 10, 11] , stresses the need for surveillance of strain diversity, to help unravel the epidemiology of this newly identified pathogen, and to provide a reference for studies into MERS-CoV evolution. All currently sequenced human and camel MERS-CoV genomes share >99% nucleotide identity across the ∼30 kb genome. Phylogenetic analysis has occurred mainly by analysing full-length genomes or multiple concatenated genome fragments, to provide reliable phylogenetic information [5, [12] [13] [14] . However, full length genome sequencing capacity is not widely available, and requires relatively high viral load, leading to limited success when trying to sequence animal or human samples [5] . An accurate typing of MERS-CoV variants, preferably with a simple assay encompassing a short region of the MERS-CoV genome, is crucial in monitoring the MERS-CoV outbreak in real time with relatively little investments on location. In this study, we describe the development of a MERS-CoV variant typing assay, which can be used in monitoring MERS-CoV circulation, especially when more information on virus type is required rapidly from a large number of viruses from animals/humans. Full or near full-length MERS-CoV genomes encompassing nucleotides 215-29770 (numbering corresponding to MERS-CoV EMC genome JX869059) were aligned with MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/) ( Table 1) . To remove the redundancy from the dataset, FastGroupII analysis (http://fastgroup.sdsu.edu/fg tools.htm) was performed grouping all currently available viral genomes based on nucleotide composition, resulting in 15 groups (Table 1) . One viral genome from each group was taken as representative in subsequent analyses. A summary of nucleotide positions that vary across the genomes was created using BioEdit v7.2.0 [15] and the number of nucleotide positions with variations was plotted over 1000 nucleotide windows. PhyML trees were generated using Seaview 4 software with the approximate likelihood ratio test based on a Shimodaira-Hasegawa-like procedure which used general time reversible as substitution model. Nearest neighbor interchange, subtree pruning, and regrafting-based tree search algorithms were used to estimate tree topologies, as described previously [5] . Total nucleic acids were isolated using an automated Mag-NAPure 96 extraction with the total nucleic acid isolation kit (Roche, Mannheim, Germany) as described previously [13] . The detection limit of the assay was determined on RNA isolated from 10x dilutions of cell culture derived MERS-CoV EMC 2012 (JX869059), as described above. Virus stocks were prepared as described previously [16] . RNA was isolated from serial 10-fold dilutions of MERS-CoV EMC 2012 [13] . Serial 10-fold dilutions of this RNA were amplified in parallel with the MERS-CoV variant typing assay described above and the upE and N gene real time PCR assays [17, 18] . The sensitivity of the MERS-CoV variant typing assay was expressed as cycle threshold value based on the upE real time PCR assay. On May 13 and 15, 2014, the first two MERS-CoV infected patients in the Netherlands who became infected upon travel to Saudi Arabia were reported to WHO; throat swabs from these patients were available [19] . In February and April 2014, nasal swabs were taken from dromedary camels of different age and sex from a slaughterhouse in Doha, Qatar [13] , which were available for this study. To identify genome regions for reliable phylogenetic analysis comparable to that of full-length genome, (near) full-length MERS-CoV genomes (Table 1) were aligned. Viral genomes that were 99.9% identical were grouped and one viral genome from each group was taken as representative in the analysis (Table 1 ) and a PhyML tree was generated (Fig. 1A) . was plotted over 1000 nt windows (Fig. 1B) . Four genome fragments, two located in ORF1a, one in S and one in ORF4b, showed a relatively high number of SNPs (Fig. 1B) and for this reason already had been used as concatenated genome fragment for phylogenetic analysis [12] . However, phylogenetic trees created for these four fragments separately did not accurately reflect the phylogenetic positions of the currently known full-length MERS-CoV genomes (data not shown). In a subsequent analysis all identified SNPs were inspected visually and regions containing SNPs with phylogenetic information regarding the previously identified four clusters of viruses were identified (Fig. 1A) . The S2 domain of the spike protein contains a number of these mutations (Fig. 1B) . A fragment of 615 bp, containing three of these SNPs, provided a phylogenetic tree similar to the one obtained upon full-genome analysis regarding the previously identified four MERS-CoV clusters (Fig. 2) , whereas other MERS-CoV genome regions did not provide similar results. As observed for the full-length genomes, human, and camel MERS-CoV genomes shared >99% nucleotide identity across the 615 bp S2 domain fragment. MERS-CoV genomes that were released recently and not taken along in the variation analysis were typed using the 615 bp S2 domain and clustered similar to their phylogenetic positions as upon full genome analysis ( Fig. 2 and Table 1 ), thereby validating the assay. A sequencing RT-PCR targeting the identified genome fragment was developed and optimized using RNA isolated from cell culturederived MERS-CoV. In limiting dilution experiments, the MERS-CoV variant typing assay amplified the 615 bp fragment down to a cycle treshold value of ∼35 as determined by diagnostic upE real time PCR assays [17, 18] . The MERS-CoV variant typing assay was used to type RT-PCR positive nose swabs from the first two human Dutch MERS-CoV cases in 2014. The results showed grouping consistent with previous findings based on long sequence fragments [19] (Fig. 2 ). In addition, the MERS-CoV variant typing assay was performed on camel samples from a slaughterhouse in Qatar [13] and sequences for 14 MERS-CoV positive animals with cycle threshold values ranging from 12.9 to 32.2 as determined by UpE real time RT-PCR [17, 18] were obtained (Fig. 2) . Five different camel MERS-CoV variants in clusters B1 and B2 were detected, without the need for full genome sequencing. Phylogenetic analysis of representative MERS-CoV full-length genomes indicated that four regions in the MERS-CoV genome exist with a substantially higher nucleotide variation across genomes. However, phylogenetic analysis of these genome regions sepa-rately did not provide reliable phylogenetic information, in contrast to an analysis of the concatenated fragments [5, 12, 19] . Subsequent analyses revealed a region in the open reading frame that encodes the spike protein with a number of positions in which nucleotide variation occurs between MERS-CoV variants with a strong phylogenetic signal regarding previously identified clusters of viruses based on full-length MERS-CoV genomes. The here- [19] and from camels from a slaughterhouse in Doha, Qatar [13] . The observed detection limit of a cycle treshold value of ∼35 allows variant typing in clinical samples obtained from humans and animals with relatively low viral loads. It enables inclusion of samples in phylogenetic analysis that would not have been included when only full length genomes would have been accepted. This MERS-CoV variant typing assay, targeting a part of the MERS-CoV spike gene, is a relatively simple RT-PCR sequencing assay that could be performed more widely as initial screening assay in laboratories with basic sequencing capacity. It provides accurate crude MERS-CoV type information, applicable in monitoring viral variants in real time. New variants identified through this initial screening could then be sent to a reference laboratory for further characterization. The continued occurrence of transmission between humans in health care and family settings is an ongoing concern as stated by the World Health Organization (http://www.who.int/csr/disease/coronavirus infections/MERS CoV Update 27 March 2014.pdf?ua=1), although the outbreaks appear to be self-limiting or extinguishable with rigorous implementation of appropriate infection control guidelines at present. However, as the primary route of transmission to humans is uninterrupted, human-to-human transmissions will continue to occur. The data obtained from the MERS-CoV variant typing assay would aid in informing the most effective international preparedness and response, allowing ad hoc risk assessment and implementation of containment strategies if necessary. 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This does not alter our adherence to all the policies on sharing data and materials. All authors contributed to gathering and analysis of the information. Saskia Smits, Bart Haagmans, and Marion Koopmans drafted and revised the manuscript based on all authors contributions. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2014.12.006.