[Molecular diagnosis and phylogenetic analysis of the first MERS case in Turkey]

Coronaviruses (CoV) are enveloped, spherical, single-stranded positive-sense RNA viruses causing mainly respiratory and intestinal infections in animals and humans. Until recently five types of human coronaviruses (HCoV-OC43, HCoV-HKU1, HCoV-NL63, HCoV-229E, SARS-CoV) have been known, however a novel CoV has been identified in 2012 in Saudi Arabia. This virus, namely MERS-CoV (Middle East Respiratory Syndrome Coronavirus), was classified within Coronaviridae family, Coronavirinae sub-family, Betacoronavirus genus, clade C. It causes acute respiratory infections in humans and transmits via respiratory route and close contact between humans. The aim of this study was to present the first MERS case from Turkey identified by molecular methods and the results of viral sequence analysis. A 42-year-old male Turkish citizen who worked as an employee in Jeddah, Kingdom of Saudi Arabia, admitted to hospital with the complaints of fever and malaise on 25-26 September 2014. Since his symptoms went on and got worse, he returned to Turkey, and hospitalized in a hospital's intensive care unit in Hatay on 6th of October with the symptoms of fever, malaise, sweating, cough and respiratory distress. He transferred to a university hospital on 8th of October and died on 11th October. The tracheal aspirate sample obtained before he died was sent to Virology Unit of Reference Laboratories of the Turkish Public Health Institution. Detection of viral RNA was performed by using a commercial real-time PCR kit (hCoV-EMC Real-Time RT-PCR, Fast Track Diagnostics, Luxembourg) targeting the MERS-CoV E protein (upE), ORF1a and ORF1b gene regions. The reference method Superscript III One Step RT-PCR (Invitrogen, USA) recommended by World Health Organization (WHO) was also applied for confirmation. Both of the methods yielded positive results for MERS-CoV RNA. For the amplification of nucleocapsid (N) and RNA-dependent RNA polymerase (RdRp) genes, hemi-nested PCR (Invitrogen, ABD) was conducted, followed by sequence analysis of 204 nucleotide part of N gene. Phylogenetic tree of N gene was obtained with the use of MEGA6 software. N gene was chosen as it comprised a two aminoacid deletion in the corresponding published sequence from the patient treated in London, United Kingdom. There was no nucleotide or aminoacid change in our isolate, namely ANK/1079/2014 when compared with human Betacoronavirus 2c EMC/2012 reference strain found in Genbank database. The target gene regions selected in our study (UpE, ORF1a, ORF1b, N and RdRp) which were also recommended by WHO, shown to have high specificity and sensitivity for the diagnosis and confirmation of MERS-CoV, and also recommended by WHO. The previous studies indicated that, the viral genomes detected in the earliest cases of humans (clade A) are genetically distinct from the others (clade B) which were isolated from dromedary camels and humans. In our study, according to phylogenetic analysis of partial N gene segment, isolate ANK/1079/2014 has taken place within clade A. In conclusion, MERS-CoV appears to have limited circulation in Arabian Peninsula and Middle-Eastern countries, it should be considered in mind that travel-related cases may export the virus outside these regions leading autochtonous infections in the other parts of the world.