Reliable typing of MERS-CoV variants with a small genome fragment.
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.


METHODS
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.
Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands; ViroClinics BioSciences BV, Marconistraat 16, 3029 AK Rotterdam, Netherlands.
Saskia L
SL
Smits
Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands.
V Stalin
VS
Raj
Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands.
Suzan D
SD
Pas
Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands.
Chantal B E M
CB
Reusken
Ministry of the Environment, Doha, Qatar; Biotechnology Research Department, Animal Health Research Institute, Agricultural Research Center, Egypt.
Khaled
K
Mohran
Supreme Council of Health, Doha, Qatar.
Elmoubasher A B A
EA
Farag
Supreme Council of Health, Doha, Qatar.
Hamad E
HE
Al-Romaihi
Supreme Council of Health, Doha, Qatar.
Mohd M
MM
AlHajri
Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands.
Bart L
BL
Haagmans
Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands; Virology Division, Centre for Infectious Diseases Research, Diagnostics and Screening, National Institute for Public Health and the Environment, Bilthoven 3720BA, Netherlands. Electronic address: m.koopmans@erasmusmc.nl.
Marion P
MP
Koopmans
D000818:Animals; D002162:Camelus / Q000821:virology; D018352:Coronavirus Infections / Q000821:virology; D016679:Genome, Viral; D006801:Humans; D065207:Middle East Respiratory Syndrome Coronavirus / Q000145:classification* / Q000235:genetics*; D058889:Molecular Typing / Q000379:methods*; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015047:Zoonoses
D012367:RNA, Viral
Camel; Diversity; Human; MERS-CoV; Surveillance; Type