1 of these papers is pretty much exactly like the other...[UPDATED #2]

This is a strange one. 

Today, a New England Journal of Medicine (NEJM) paper [2] came out and received a vast amount of media coverage. 

It seems as if all the other recent press, the camel kissing videos, camel advocates decrying the link between MERS-CoV and their beauties and camels being included in risk assessments...have just primed the world for the next scientific paper. 

And then this new paper came out showing MERS-CoV infection of a farmer and of his camels and a likely direction to that infection of camel >> farmer. And, it came out in the highly prestigious NEJM - this folks, is one part of what a high impact factor is all about - wide exposure and broad coverage. You really get your research out there.

Apparently neither of us are actually
Jeddah camels 'cause we're wearing too
much bling.

Twitter and the mainstream media have lit up with lines like "direct evidence that MERS comes from camels", "new report offers strongest evidence yet that MERS virus spreads from camels to people", "1st evidence that a new deadly virus has been transmitted from a camel to people".

There is a problem though. It's not reeeally any of those things.

These are both studies of what looks to be the same infected human (a 43-year or 44-year old man depending on which report), hospitalised at King Abdulaziz University Hospital in Jeddah on 3-November-2013, owner of a herd of 9 camels, some of whom were sick, sampled at around the same time (I presume, otherwise why sampled at all?).

The only major differences (there are smaller differences) between the 2 reports is which camel yielded sequence - it seems to have been Camel G for Drosten's lab and Camel B for Madani's - they even seem to have used the same identification scheme for the camels! The NEJM paper also has some human serology data that were absent from Drosten's study; determined using an immunofluorescence assay, although not confirmed as MERS-CoV specific using the more specific neutralization test.

Oh, one other big difference.

Memish, Drosten and colleagues got their paper out online around 20-March-2014 (2-months 15-days earlier [UPDATED]).

But let's also look at the sequence release/modification date too. This is the date when the researcher's virus sequence data, submitted to the public sequence database GenBank prior to the paper being published, is available. For Azhar and Madani and colleagues, that date was 1-May-2014 (sample taken 5/8-Nov-2013) and for Memish and Drosten and colleagues, 24-March-2014 (could only access 3 fragments; sampled 9-November-2013).

NOTE: This Editor's Note was added (9-June) to the NEJM paper:

The patient and camels discussed in this article are also described in Memish ZA, Cotten M, Meyer B, et al. Human infection with MERS coronavirus after exposure to infected camels, Saudi Arabia, 2013. Emerg Infect Dis 2014;20:1012-5.

I'm sure there are at least 2 very interesting stories behind this little event.

References...

1. Human Infection with MERS Coronavirus after Exposure to Infected Camels, Saudi Arabia, 2013
   Ziad A. Memish, Matthew Cotten, Benjamin Meyer, Simon J. Watson, Abdullah J. Alsahafi, Abdullah A. Al Rabeeah, Victor Max Corman, Andrea Sieberg, Hatem Q. Makhdoom, Abdullah Assiri, Malaki Al Masri, Souhaib Aldabbagh, Berend-Jan Bosch, Martin Beer, Marcel A. Müller, Paul Kellam, and Christian Drosten
   Emerging Infectious Diseases
   http://wwwnc.cdc.gov/eid/article/20/6/14-0402_article.htm
2. Evidence for Camel-to-Human Transmission of MERS Coronavirus
   Esam I. Azhar, Ph.D., Sherif A. El-Kafrawy, Ph.D., Suha A. Farraj, M.Sc., Ahmed M. Hassan, M.Sc., Muneera S. Al-Saeed, B.Sc., Anwar M. Hashem, Ph.D., and Tariq A. Madani, M.D.
   New England Journal of Medicine
   http://www.nejm.org/doi/pdf/10.1056/NEJMoa1401505

17 new MERS-CoV sequences bind perfectly to frontline screening PCR assay for MERS...

Click to enlarge. The primers/probe are depicted as grey boxes.
If mismatches existed they would show up as horizontal black
lines within the grey box. No mismatches are evident.
The GenBank accession numbers are
shown on the left of this alignment of 17 MERS-CoV
sequences.

Only 17 of the 45 sequences seem to include the region covered by the upE laboratory assay I just posted about in the WHO laboratory testing update but of those, the forward and reverse oligonucleotide primers and the probe all bind without any mismatch.

While that may sound like an obvious statement considering that these viruses were probably detected using that assay it isn't.

The new MERS-CoV sequences were determined using using unbiased 2nd generation high-throughput sequencing technologies that did not rely on these primers to generate them. So we are now able to check and see if there are any nucleotide changes at the target sites for the primers and probe, that would reduce the efficiency the assay.

There are no such oligonucleotide mismatches between primer and viral genes among those 17 sequences, which is good news for that assay's continued usefulness.

Built to last eh?

T.perforatus MERS-CoV strain sequence, and others, online...

If you're a bit of a sequence collector/hoarder/nut then you'll be interested to know that the recent bat CoV RNA-dependent RNA polymerase (RdRp) sequences are now online on GenBank.

• Bat coronavirus Pikuh/CII_KSA_001/Riyadh/Saudi Arabia/2013
• Bat coronavirus Rhhar/CII_KSA_002/Bisha/Saudi Arabia/2013
• Bat coronavirus Rhhar/CII_KSA_003/Bisha/Saudi Arabia/2013
• Bat Coronavirus Taper/CII_KSA_287/Bisha/Saudi Arabia/2012
• Bat Coronavirus Rhhar/CII_KSA_004/Bisha/Saudi Arabia/2013

These seem to include the primer regions judging by their length. Consider that when using them.

The MERS-CoV strain from T.perfortaus is CII_KSA_287 - highlighted in bold. Please note, that at writing, it is erroneously identified on GenBank as originating from Rhinopoma hardwickii. It should be Taphozous perforatus.

Tracking MERS-CoV through time: a spikey problem

This morning on Twitter, Helen Branswell (@HelenBranswell) asked this question, with a comment...

How is this so? 16 months after the 1st known #MERS cases, there are only 9 viral sequences in the public domain. So much for collaboration.
— Helen Branswell (@HelenBranswell) August 6, 2013

So I thought a little perspective might be nice. 

The SARS epidemic had its origins around Nov 16th 2002, although the major activity started in Feb of 2003. 

• 64 human SARS-CoV genomes had been produced by September 2003 ([UPDATED:] see Science paper). That is by 317-days later, or 10-months, 13-days (perhaps less given that the genome sequences were possibly sequenced well before the paper was submitted e.g. late phase genome s seem to have been submitted to GenBank by July 2003). 
• For MERS-CoV we currently have 9 genomes at 505-days (give or take), or 1-year, 4-months.

Not that anyone needs to be reminded, but 80% of MERS-CoV cases come from the Kingdom of Saudi Arabia. The world is relying on them, or their collaborators, to turn the nucleic acid extracts used to define these cases (PCR-POSs hopefully kept in a -80'C freezer), into templates for gene or genome sequencing.

I personally don't believe we need to have complete genomes right now in order to fulfil the fairly urgent public health need to monitor the virus and notice if it changes, or is changing, or is not changing. These changes tell us whether the virus is still adapting or has settled in - perhaps having done so prior to this outbreak's indicator, severe disease. 

What else to use to track adaptation?

Perhaps the 4,000nt Spike (S) gene, or some smaller but suitably variable portion of it, could be a target for sequencing? 

Zhang and colleagues have data showing it could be used to track an animal coronavirus's adaptation to humans, through its 3 pandemic phases. This was done using phylogeny (a way to show how one sequence relates to another through time and space) of nucleic acid sequences and alignments of the translated version of these sequences. All we need is primer sequences that could be used to reliably amplify the S gene of the MERS-CoV. If anyone has those already perhaps they could publish them...if they haven't already. A very brief look at the 9 MERS-CoV genomes already shows some variety. Perhaps unsurprisingly, there is very little change among the 4 Al-Ahsa genomes; their collection dates are separated in time by 17-days.

This shows a schematic of the aligned Spike genes. The black lines within the grey boxes represent nucleotides that differ from the consensus. More differences are obvious in the earlier sequences. The oldest MERS-CoV isolate is at the bottom, the most recent, at the top (detailed below). See the full version here at VDU.

Interestingly, the phylogeny of the complete Spike genes looks  similar to that of the complete MERS-CoV genomes. However  its doe snot place the isolates in order of increasing time to the extent that the full genomes do. I also looked at a 900bp fragment of the 3' of the Spike gene - easier to amplify but a very similar tree to that of the complete Spike.

All 9 complete MERS-CoV spike protein genes (nt). Alignment in Geneious Pro, tree in MEGA 5.10.
Full version will be here at VDU.

All 9 complete MERS-CoV genomes (nt). The arrow indicates moving forward in time; the oldest MERS-CoV isolates at the bottom, the most recent at the top. Alignment in Geneious Pro, tree in MEGA 5.10.
Full version will be here at VDU.

So where does that leave us?

Adaptive pressures on the SARS-CoV drove its genome towards settling down in the late stage of the 3-phase outbreak (defined by the Chinese SARS Molecular Epidemiology Consortium), with changes in the Spike gene occurring before that. Complete genomes are clearly the gold standard - so I dial down that personal belief from earlier.

The Spike gene still seems a useful target for MERS-CoV too, although not as accurate at plotting the time of virus isolation as complete MERS-CoV genomes were in my example above. Still, it, or some part of it, is still of use as an early-warning system to alert us to viral change and it will prove easier to amplify by smaller or less genomics-focussed laboratories. Something we need to consider in order to get some information, which is far better than none.

While we've seen predictive modelling for the age of MERS-CoV, we don't actually know when the virus came to be or when it started spilling over to humans. More full genome sequences would certainly help address that question. And finding its origin.

However, perhaps we should make the trade off and use the 3' end of the Spike gene now, in an effort to keep some sort of eye on how the MERS-CoV is travelling? Anyone else have a good region that fits the bill?