Novel human Coronavirus MERS-CoV

As announced by the Coronavirus Study Group, we use the virus designation "MERS-CoV" where "MERS" stand for "Middle East Respiratory Syndrome".

Therefore the preliminary name (HCoV-EMC) was replaced.

The primary isolate was obtained in cell culture by Dr Ali Zaki, the first author of a study published in the New England Journal of Medicine describing the virus.

The Department of virology at the university of Rotterdam, The Netherlands - characterized the virus initially.

download the paper by Zaki et al.

 

Our diagnostic laboratories accept samples for diagnostic investigation of cases compatible with the WHO case definition. We are also providing reference services (i.e., confirmatory testing and troubleshooting) to laboratories worldwide.

Link to the current WHO case definition

Link to the WHO interim recommendations (September 2014) "Laboratory Testing for MERS-CoV"

 

Detecting MERS-CoV by PCR and sequencing

Real-time RT-PCR assays and RT-PCR assays for sequencing

We have developed and validated a set of RT-PCR assays for the detection of MERS-CoV. This is the test recommended by WHO. We provide control material necessary for the conduction of the test to laboratories worldwide. Please consult the information below, and please read the Discussion section of the articles for important recommendations. 

download our first article describing the WHO-recommended test for MERS-CoV

In this article we present two real-time reverse-transcription polymerase chain reaction assays for MERS-CoV, targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b (27 September 2012).

 Link to our publication about assays for laboratory confirmation of novel human coronavirus (MERS-CoV) infections

In this second article we present a highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described (RdRpSeq assay and NSeq assay). Finally, a protocol for detection of antibody response by immunofluorescence microscopy was developed (06 December 2012).

We provide here bench protocols that summarizes the formulation of the MERS-CoV test in a practical way:

Bench protocol (upE and Orf 1b assay) download here.

Bench protocol (1A assay) download here.

Bench protocol (RdRpSeq and NSeq assay) download here.

download sequence alignments (as zipped *.fas files) comparing the results of RdRpSeq and NSeq sequencing assays. The sequences obtained from a patient from Essen, Germany, the HCoV-EMC/2012 and the London sequences are included.

Positive control
We provide positive control material (for the upE assay and the 1A assay) to qualified laboratories worldwide. The material is a small fragment (< 600 bp) of synthetic RNA representing the part of the viral genome that is detected by the real-time PCR assays we developed. We notify WHO of those entities that have received the material. In order to keep the required administrative work to a manageable level, the ordering and shipping data are handled through the Europan Virus Archive (EVA), a non-commercial entity funded by a European Union infrastructure grant. Please address your request via the EVA homepage. EVA will immediately notify us and we will initiate shipment as soon as possible. The material is shipped from our institution in Bonn/Germany. A small non-profit charge applies for the shipment. Exemptions are possible. More information...

Link for ordering the "upE assay" positive control 

Link for ordering the "1A assay" positive control

download results of both real-time RT-PCRs (upE and 1A assay) on serial dilutions of the corresponding positive controls and the sequences of both controls (*.pdf file).

 Oligonucleotides
Oligos (=primers and probes) as published can be retrieved from stock at Tib-Molbiol, Berlin. The ordering of these reagents from stock provides the advantage of receiving reagents from a large synthesis lot that has been quality-tested on actual virus material in our laboratories, while the oligonucleotides themselves have not physically passed through our facilities. This excludes any possibility of reagent contamination.
More information...

 

Serological testing for MERS-CoV

Recommendations regarding serological testing

No official recommendations are currently available regarding serological tests. The following recommendations are based on our own experiences and should be regarded as a personal opinion and statement by C. Drosten and M.A. Müller. For more detailed information on serological testing and evaluation please refer to:

Drosten et al. New England Journal of Medicine 2014 "Transmission of MERS-Coronavirus in Household Contacts"

Please also read the latest interim WHO recommendation (revised in September 2014):

Link to the WHO interim recommendations (September 2014) "Laboratory Testing for MERS-CoV"

 

We are currently recommending a two-step diagnostic approach:

Step 1. Recombinant enzyme-linked immunosorbent assay  (ELISA)

Substrate: purified recombinant spike protein (subunit 1 domain) of MERS-CoV

Testing material: Human serum, diluted 1:100

Diagnostic parameter: Immunoglobulin G (Ig)

Alternative to ELISA:  Conventional immunofluorescence assay (cIFA)

Substrate (glass slides): MERS-CoV infected Vero cells mixed 1:1 with non-infected cells (inactivated)

Testing material: Human serum, diluted 1:40 /1:100

Diagnostic parameters: Immunoglobulin G (Ig)G and IgM in patient serum

Step 2. A) Recombinant IFA (rIFA) using spike protein

Substrate (glass slides): Transfected Vero cells expressing recombinant spike protein of MERS-CoV. Remark: For differential rIFA prototype humanpathogenic CoV (HCoV-NL63, -229E, -OC43, -HKU-1, SARS-CoV) should be included.

Testing material: Human serum, diluted 1:40

Diagnostic parameters: Immunoglobulin G (Ig)G and IgM in patient serum

B) Confirmation by virus plaque reduction neutralization test (PRNT)

Substrate: Vero B4 cells and infectious MERS-CoV strain EMC (requires BSL3 laboratory)

Testing material: Heat-inactivated serum/ dilutions starting at 1:10 until 1:1,280

Diagnostic parameter: MERS-CoV neutralizing Ig in patient serum. According to our experience a 50% plaque reduction at a serum dilution of 1:20 can be rated positive.

Important information: The use of the mentioned serological methods is only to be applied in individuals with symptoms according to WHO case definition and a history of proven exposure to a laboratory-confirmed case of MERS-CoV during the infectious period.

We provide rIFA slides in limited quantities to qualified laboratories - please contact us (muller(at)virology-bonn.de). Larger quantities of the recombinant ELISA and conventional IFA slides can also be ordered via EUROIMMUN (contact: Dr. Erik Lattwein, e.lattwein(at)euroimmun.de, EUROIMMUN Lübeck, Germany).

MERS-CoV provides a fine granular cytoplasmatic fluorescence pattern sparing the nucleus. Examples are provided in the paper and in the bench protocol referred below. Any sign of reactivity must be confirmed in consultation with a reference institution before any further steps toward notification are taken.

It is important to know that IFA will generate false-positive results due to cross-reactivity between anti-HCoV antibodies. It is impossible to discriminate cross-reactive antibodies by IFA.

We provide reference services for serology. We re-test reactive sera tested by conventional IFA or other serological assays (e.g. ELISA, Western blot analysis) in differential rIFA and plaque-reduction neutralization tests for confirmation. These highly specific tests are less cross-reactive but cannot be shipped to other laboratories. Please contact us. We will provide rapid assistance wherever possible.

 

  Link to publication about assays for laboratory confirmation of novel human coronavirus (MERS-CoV) infections, including suggestions for confirmatory testing by serology.

Bench protocol: Conventional immunofluorescence assay (cIFA)

download here.

Bench protocol: Recombinant immunofluorescence assay (rIFA)

download here.

 

Our work on detecting and diagnosing emerging viruses is supported by EMPERIE, an EU FP7 project More information...

Our applied and basic research in related fields is supported by the German Ministry of Education and Research (BMBF), as well as the German National Research Council (DFG). We are a part of the German Centre for Infection Research (DZIF).