Dutch researchers in collaboration with Qatar are at work sequencing MERS-CoV from camels...

And from the WHO comes confirmation of some of my earlier bits and pieces about the MERS-CoV in camels story from earlier....

The three camels which have tested positive with #MERS in #Qatar were detected in a barn linked to two confirmed human infections
— WHO (@WHO) November 28, 2013

Further, some very interesting titbits from a Twitter exchange this evening.

Firstly Prof. Marion Koopmans, Head of Virology at the Laboratory for Infectious Diseases of the National Institute of Public Health in the Netherlands confirmed that this was the MERS-CoV and not something requiring lengthy sentences filled with "probable" and "MERS-CoV-like"...

@MackayIM working on it. Qatari's are fast, samples came in last weekend, but yes, confirmed
— Marion Koopmans (@MarionKoopmans) November 28, 2013

..and that for the most useful conclusions to be drawn from any sequencing being undertaken..

@arambaut @mackayim Yes, but requires sufficient sequence of sufficient quality. We are working on it
— Marion Koopmans (@MarionKoopmans) November 28, 2013

..but that despite all sorts of great leaps in technology, not to mention in distance-spanning scientific collaborations, things don't just happen overnight. 

We should all be mindful that there are many steps between taking a (hopefully adequate) sample(s) from a human or animal, and reaching any useful conclusion about how the molecularly characterized virus might have travelled (human to dromedary, vice versa or via some other vector or intermediate)...

@arambaut @mackayim Yes, but we DO need some time. We are 4 days into the labwork. ;>)
— Marion Koopmans (@MarionKoopmans) November 28, 2013

As Prof Andrew Rambaut, Institute of Evolutionary Biology, University of Edinburgh, noted...

@MarionKoopmans @mackayim Indeed - I don't think people realise how much work it is to sequence these (and how much work to get good seqs).
— Andrew Rambaut (@arambaut) November 28, 2013

And on the subject of whether the new sequences will lead to an indication of which direction this particular cluster of infections is travelling i.e. from human-to-camel or camel-to-human, Prof. Rambaut had this thought on following the viral genome's sequence variations (polymorphisms)...

@MackayIM @marionkoopmans Look for polymorphisms that are not fixed in the putative donor but fixed in the recipient...
— Andrew Rambaut (@arambaut) November 28, 2013

This is all really great to watch. A fast and fruitful collaboration between sample holders and laboratory researchers, expert in their fields.

Click on image to enlarge.
Those POS for a fragment of MERS-CoV or
MERS-CoV-like virus sequence are highlighted
in red. Whether there are other intermediates
remains to be confirmed.

At this point, I believe (and it is just a belief) that the camel is looking good for a source of MERS-CoV acquisition by humans. Is it an endemic camel virus? Well, we still have the knowledge that bats seem to harbour a lot of CoVs, and there is that pesky Taphozus perforatus sequence discovered from earlier in the year. It looked an awful lot like a fragment of the MERS-CoV genome. Baboons - I'm holding out for them to be the link between bats and camels...but that is a hope in the absence of any data whatsoever!

Today's confirmation of a cluster of 3 POS camels among 14 represents 21% of the animals POS in a single area. 

If we consider this to be human-to-camel transmission, then this would be a much steeper proportion of positives than we normally see when we look at studies of close contacts of human MERS cases. Camels must be very susceptible to MERS-CoV infection because human contact testing just does not show this level of onward transmission. More susceptible to humans? No, I think we're getting closer to confirming that it's a camel-to-human thing...but we are not there yet.

Work continues, but today was a significant day and one in which I give thanks for the ability of people from all over the world to work together towards common goals in preventing human disease. 

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?

An entire H7N9 genome from a clinical specimen in one shot

Some weeks back, Ren and colleagues described their use of next generation sequencing (or "deep" sequencing; unbiased, massively redundant sequencing of all the DNA or RNA in a sample - to put it very basically) to pull out the entire genome of an influenza A(H7N9) virus from 1 clinical sample.

The new genome is called A/Jiangsu/2/2013(H7N9) and can already be found on GenBank. It's 8 segments are numbered in order from KF226105 to KF226112.

The authors found the E672K (PB2 gene segment) and I368V (PB1) mutations related to virulence and transmissibility

The caveat for this sequencing, in case you're making comparisons to the recent small sequence fragment obtained from the MERS-CoV strain detected in bat poo pellets, is that they had enough fresh human sputum (probably not freeze/thawed and left at room temperature as with bat samples) from the 54-year-old female case from Zhejiang to be able to purify, concentrate and clean up the virus before preparing the nucleic acids for NGS. That likely means a lot more virus, from a more hospitable environment for virus, and a lot less other nucleic acid and junk to interfere with the amplification and sequencing components.

Interestingly the authors found that in 22 nucleotide positions, more than a single nucleotide could be reliably identified - sequence heterogeneity that shines a light on how much viral change goes on, even within a host, as the virus keeps testing out different ways to replicate, bind and enter cells and interfere and avoid the immune response against it. Some of those changes altered the protein (amino acid) sequence.  Sometimes those changes are no good for the resultant virus and the strain with that/those changes replicate poorly, sometimes there is no change in replication or infection efficiency and sometimes the process generates a more efficient viral strain that may then grow to become the dominant strain which we cough and sneeze onto our contacts. 

This unbiased (doesn't rely on a very specific PCR, and the amplification of the most dominant sequence in a sample as do traditional Sanger sequencing methods  sequence analysis approach is a great way to find those changes in a hole-of-genome approaches - because it's likely that "favourite mutation X, Y and Z" only represents a portion of the changes that go into making more successful influenza viruses.