Laboratory investigation and phylogenetic analysis of an imported Middle East respiratory syndrome coronavirus case in Greece

Abstract

Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1st and 2nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient's history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract.

Fig 1. Phylogenetic analyses of the MERS-CoV S and N ORFs.

Midpoint-rooted phylogenetic trees of the full-length nucleocapsid (N) and spike (S) open-reading frames (ORFs) obtained from the clinical sample and published nucleotide sequences available from i) GenBank, ii) the Health Protection Agency (HPA) website (http://www.hpa.org.uk/webw/HPAweb&HPAwebStandard/HPAweb_C/1317136246479) and iii) the Institut Fr Virologie (IFV) website (http://www.virology-bonn.de/index.php?id=46). The estimated neighbor-joining trees were constructed from nucleotide alignments using MEGA version 6.06. Sequence names are derived from Genbank accession number|virus strain name|month-year of collection. Numbers in the parentheses denote additional human sequences identical to the listed sequence. An asterisk (*) denotes 2 additional identical N ORF sequences obtained from IFV, including strains MERS-CoV/Jeddah_2014_C8826 and MERS-CoV/Jeddah_2014_C9055. Two asterisks (**) denote 3 additional identical S ORF sequences obtained from IFV, including MERS-CoV/Jeddah_2014_C9055, MERS-CoV/Jeddah_2014_C7770 and MERS-CoV/Jeddah_2014_C7149. MERS-CoV sequences derived from camel specimens indicated by camel icon. Bootstrap support values (1000 replicates) 75% are plotted at the indicated internal branch nodes. Scale bars show the number of nucleotide substitutions per site.