Sunday, 25 August 2013

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.