Is there a better smoking bat or camel?

That teensy fragment of Middle East respiratory syndrome coronavirus (MERS-CoV) sequence (yes, I called it a fragment of that virus) from a Taphozous perforatus bat caused a lot of hassle last week,certainly a disproportionate amount to it's representation of only 0.5% of a MERS-CoV genome. Similarly, the report of MERS-CoV protein-reactive antibodies in camels some weeks back.

In a New York Times (NYT) article discussing the discovery of the 180-203 nucleotide (nt) gene fragment in a Saudi Arabian tomb bat, Donald McNeil opened with..

Health officials confirmed Wednesday that bats in Saudi Arabia were the source of the mysterious virus that has sickened 96 people in the Middle East, killing 47 of them.

The size range represents numbers used in various articles and of the fragment from the public sequence database GenBank (203nt) -"~190 nt" noted in the actual scientific publication). By the way, has so much ever before been written about so tiny a sequence?

Because he did not include in this line, or his article, a list of all the various possible scientific shortcomings, he didn't write in more detail about the difficulties with linking a virus in any sample to the cause of a disease  in humans, he forgot to specify that this was not the actual virion that caused MERS in the human index case in Bisha, he left out the PCR-101 section on why detecting a genetic sequence is not the same as isolating an infectious virus or how to interpret a PCR fragment's sequence....he was, in some circles, criticised. 

Okay, so the bat study did not isolate infectious virus, could not obtain any other sequence, found sequence in a bat from the family Emballonuridae rather than the "expected" Vespertilionidae bats and the positive sample was from bat faeces rather than blood or some other sample more convincing. Perhaps insects, food for T.perforatus, carry MERS-like CoVs? No-one has ever found that though. Is there evidence for 2 CoVs to be completely different except for a stretch of ~100% identity? Don't know, but don't think so. As Prof Andrew Rambaut noted in his very detailed analysis of this fragment, it does differ by 1 nucleotide from many MERS-CoV sequences (so its 99.5%-100% identical). Is there a more likely animal carrying a more similar MERS-CoV strain of virus that is spilling over to humans causing MERS cases? None that has been publicised to date. And therein likes my beef with some of the criticisms I have read this week. 

So far, this finding is the best lead we have in finding an animal source. There is no evidence to dispute the link, any more than there is evidence to prove it. Yes, there may be a another smoking bat or camel or something else out there. But it hasn't been found yet. And the public might like to hear how researchers are progressing rather than wait the very long time it will take to dot Is and cross Ts on the final MERS-CoV life-cycle, once they determine it.

And by the way, there is no other CoV sequence on GenBank that has >90% nucleotide identity with the T.perforatus sequence, except for the human MERS-CoV sequences. That doesn't exclude there being some other recombinant or novel CoV out there, but that is pure speculation; more so than saying that this new sequence represents a strain of the MERS-CoV found in humans.

In succinctly summarising some of the criticism, an article in CIDRAP presents a great overview and hints at what this criticism implies; that stories in the media must get every detail spot on or the writer may be portrayed as a poor scientist.

What? Wait. Seriously??

This was a newspaper article in the New York Times people. It was about 870 words long. It won't be setting global health policy nor will it be creating a WHO disease notification stating Taphozous perforatus bats, in particular, are the primary source of all MERS cases. Or of any cases. I don't doubt this is a prestigious newspaper but this story will likely be nest week's fish 'n chip wrappings (does anyone still use newspaper to wrap fish 'n chips? Is there a digital equivalent of - "yesterday's homepage, tomorrow's archive"?). In my opinion, and I don't mean to speak for all, scientists and health policy makers know that a newspaper is not a peer-reviewed scientific journal and that it's intent is to inform it's readers so they'll come back.

Were the many readers of the NYT misinformed? Perhaps the "health officials" could have been better defined by the NYT article. Presumably it's the authors-researchers may have been a better descriptive (as was used in a follow-up piece), probably more in tune with the public's perception of us. Beyond that the article did a good job of presenting the results of a research paper's relevant findings to the wider audience. They also both caution against over-interpreting the data. The NYT article has that well covered; a transmission route is not clear, more testing needed, more work being done, sample degradation due to a break in the cold chain, it was only 1 bat.

I very much agree with comments in Robert Roos CIDRAP article; the critics are getting carried away. There are many different levels of science communication that reach the general community - the popular press are not Lancet, and vice versa.

If you really want to pick holes in a part of the coverage, you might well ask why so many are looking at a 180-190nt fragment when the ends of that PCR-amplified fragment actually reflect the commercially made oligonucleotide "primers", not the (likely) viral template at all.  The actual fragment that should be analysed from the T.perforatus bat droppings is, at best, 156nt (but only 137nt if the internal nested PCR product was sequenced but the product on GenBank, 203nt long, includes both internal and external primer sequences in it-PCR speak here, sorry). Probably won't change the outcome of any analyses to date (156 still encompasses the nucleotide variation and is still differs by 11% from any non-MERS-CoV sequence), but it is a different number. 

I'm sure a newspaper headline "156 nucleotides of a 30,130 nucleotide genome possibly related to the mystery virus that may have directly or indirectly killed 47 people in Saudi Arabia" would be a real page turner.

So, let's keep up the good work of presenting and trying to deconvolute our own studies, let's keep the public interested and informed without overcooking the message, let's allow for imperfection (as readers of this blog will be all too familiar with), but perhaps let's keep the peer reviews to the scientific literature where they are in demand and required...and keep perspective on these new findings when they come to our attention through the popular press.

Why only 181 nucleotides of T.perforatus MERS-CoV sequence?

In some of the many articles written about the new discovery this week, there were comments along the lines of  its amazing any sequence could be obtained from the samples cause they had sat for 48-hours at US customs and thawed. A more precise quote could be found here for example.

I have some thoughts on that - and these come from me, someone who has worked with a lot of clinical human specimens from which I've been able to amplify viral bits and pieces on a regular basis. Many small (200-600 basepairs[bp] fragments) but also longer pieces of >1,000bp, assembling small viral genomes from them. These samples may >10-years old, having been freeze-thawed numerous times after spending various amounts of time in courier vans, planes or sitting at room temperature before having nucleic acids extracted, tested and eventually (extracts may also sit around during testing and preparation and be freeze-thawed etc) frozen at -20°C or -80°C.

Keeping in mind that this issue of thawing might simply be a case of "hold your horses people". The EID paper was an early and quick report announcing the discovery of this MERS-CoV strain. So, my thoughts:

1. Because the materials that yielded the sequence (collected in October 2012) were described as "thawed" we can presume that the dry ice they were shipped with ran out during the transport to, or waiting time at, US customs. Once the refrigerant is all gone, the samples would come to room temperature as fast as the cardboard box and plastic receptacle it held, allowed. The publication described them as having been thawed for 48-hours.
2. How warm are we talking? The average temperature of Bisha (where the Taphozus perforatus bat was found, in an old date orchard outdoors) in October ranges from 15-20°C to 30-35°C. I don't know where the US customs site was so don't know that temp range - but expect it's less. So let's make some wholly unfounded assumptions:
• That this MERS-CoV strain can spread via the virus found in faecal pellets or other bat excreta. Perhaps as wind-blown dust or to other animals via a faecal-oral route. Even if the bats are hanging from a cave ceiling, but certainly when they are hanging outdoors, the virus must be capable of surviving in faecal pellets at a very high "room temperature" to complete a transmission event. If they can survive, that means intact virus - RNA genome + proteins + capsid + lipid envelope - the whole lot. For RT-PCR - you only need the RNA bit, not infectious virus. So, you're already lowering your expectations for what's required of a "successful" shipment.
• To confirm bat species, a genetic test was used which required the amplification of another piece of DNA - a region of the cytochrome B gene was amplified and sequenced. How large this fragment was, I'm not sure. However, a relatively large fragment of this gene can be used to differentiates bats, useful when you can't tell them apart by looking at physical features. Other work on opossums by the collaborator who helped sequence this region (Dr George Amato) in bats, employed >800bp of sequence. Why did this fragment amplify so well if the viral RNA did not? Perhaps because DNA is more hardy (various reasons) or because the bat blood or skin that it was amplified from, better protected the DNA from the thawing than bat faeces did for the viral RNA? Or...
3. Perhaps the primers used for other regions of the T.perforatus MES-CoV strain failed because the virus was too genetically distinct. I've had a look at the alignments and the primer binding sites can be found so it's probably not that. However, some of these primers that produce larger products are very degenerate (primers specially designed to account for nucleotide variation in a range of subtly different viruses or viral strains). 
• Degenerate PCR primers generally have much decreased sensitivity compared to 100% target-specific primers. This drop in ability to detect low amounts of RNA is the case even when using nested PCR - sorry if this has become to PCR technical! 
• The primers that did work for the T.perforatus bat MERS-CoV, Nested CII-MERS-RdRp, were much more target specific with only 1 degenerate base in 4 primers. That, combined with a drop in viral RNA amount, may well be why this 1 assay worked, worked where the others did not.
4. There was no mention in the EID paper of the use of an internal control RT-PCR target - a region of a gene in bat faeces (or blood or tissue depending on what was tested) that might allow some quality monitoring to see if there was truly decreased amounts of intact RNA in the October 2012 batch compared to that in the April 2013 batch of samples. That would be helpful to know which course to follow next.

So what does all this mean? Just me thinking in print I guess.

It's always important to maintain the cold chain from sample collection through to nucleic acid extraction and template addition to an RT-PCR/PCR tube. But I think we should look elsewhere for reasons why the T.perforatus MERS-CoV-positive sample has not yielded more than 1 fragment from the few assays used. 

I wouldn't be surprised if there was more sequence coming soon from this sample.