Monday, 18 November 2013

Should I have published?
Since finishing my PhD I've been faced with a dilemma. In a nutshell, having come across a (somewhat serendipitous) observation in my PhD studies, should I publish it? I decided to publish, and it was both an editor's pick and is now regarded by the journal as 'highly accessed' (the importance and possibly ephemeral nature of such labels is a completely different discussion). That implies it was worthwhile, but was it?

The study revolved around an observation in BHK (hamster) cells infected with Bluetongue virus (BTV). The cells looked very strange: rounded and with condensed DNA/chromosomes in a pattern suggestive of some stage in mitosis, albeit a rather odd looking mitosis. To try and see what's going on, we used confocal microscopy with a panel of antibodies to look at the status of various parts of the cell division machinery. In brief, we found that the centrosome, a major orchestrator of mitosis, was severely disrupted. Co-incidence or not, the BTV protein non-structural protein NS1 also located in the region.

A-D. Different BTV serotypes (16, 1 and 8) induce aberrent mitoses (although BTV-16v induces the most). Different cell types can also be affected, although BHK cells appeared to be the most susceptible.
Something that was conspicuous was the association of the viral NS2 protein with the condensed chromosomes. When we took a series of images in the z plane and analysed them it became clear that NS2 appeared to be associated with the kinetochore. Combined with the observation of its location on microtubules, it is conceivable that NS2 may be a microtubule cargo molecule (or interacting with one) that obscures the kinetochore during the initial stages of mitosis. As the microtubules polymerise though the cell, the tips don't find the kinetochore, resulting in faulty mitosis. Many viral proteins use microtubules to get around and, based on other viruses, the dynein/dynactin complex would be an interesting  place to start looking for a protein that interacts with NS2.

A. NS2 expressed from a plasmid locates to microtubules (red). B. Z stack images reveal NS2 located at positions suggestive of the chromosome centromeres. C and D. Expression of NS2 from a plasmid recreates the aberrent mitotic phenotype.

To look at whether NS2 alone is capable of inducing the aberrent mitosis, we transfected cells with plasmids encoding the protein. When looked at from a confocal perspective, the transfected cells appeared to reflect the phenotype seen with virus infection. When a GFP-tagged version of NS2 was used in live cell imaging, we found that the cells were less likely to complete mitosis correctly, spent longer in mitosis, and resulted in an increased level of binucleated cells.

Transfecting HeLA cells with a palsmid expressing a GFP-tagged version of BTV NS2 resulted in a longer time spent in mitosis, a reduced level of successful mitosis, and binucleation.
 So, to the options. 
1) don't publish. At the end of the day it's just an observation; I have not elucidated an exact mechanism and nailed down a precise protein, as would be expected for a publication in a journal of greater 'impact'. Not taking the story to an end, followed by publishing in a prestigious journal might be viewed as poor science by some. 
2) publish. Many would argue that publishing information, regardless of how seemingly insignificant, is important and, arguably, a necessity based on the fact that it is being funded by the public.

I published. Partly for the reasons outlined in scenario 2, but also because the study was at a point where other people had contributed work, in which case it would not be fair for them to have done this work only for me not to publish. Of course, continuing the project to the end would have been my (and my collaborators') preferred option, but time ran out. As it stands, this observation is in the public domain for all to see, with the option of progressing it further to try and unravel what's happening.

Should I have published? I'm satisfied that I did, but it once again highlights the question of how many other such observations are languishing in abandoned lab books around the world.

Andrew E Shaw, Anke BrĂ¼ning-Richardson, Ewan E Morrison, Jacquelyn Bond, Jennifer Simpson, Natalie Ross-Smith, Oya Alpar, Peter PC Mertens and Paul Monaghan (2013). Bluetongue virus infection induces aberrant mitosis in mammalian cells Virology Journal DOI: 10.1186/1743-422X-10-319

Sunday, 10 November 2013

Down on the farm with Schmallenberg virus: the full story
I've already written a couple of posts about Schmallenberg virus (SBV), a bunyavirus that emerged in Northern Europe in 2011. What I haven't discussed is the SBV experience on my family's diary farm. Clearly an opportunity not to be missed, this has just been published. The thing that scientific publications can't convey however is the meandering thoughts and subjective observations that have little or no scientific rigour. A scientific manuscript can only report concrete and measurable results. Hence this post.

SBV is difficult to spot as the most dramatic clinical signs tend to be malformed offspring, which is a somewhat rare occurrence, at least in cattle herds. As a result there is a period whereby the virus may have been around for several months before it is discovered: when we first found SBV on the farm, the UK was more or less at this stage. In February 2012 there were only a few reported cases of SBV, all around the SE fringes of England where SBV-laden Culicoides had presumably been blown across the channel.

No doubt at least partially as a result of my ongoing comments on the phone about the SBV situation, when a cow oddly aborted close to term, "could it be SBV?" was a question that immediately arose. The cow, number 157, along with some others, was bled and the samples sent to me in Glasgow, where I tested it for SBV antibodies. The result was a clear positive for SBV.

The first ELISA result of SBV on the Bishops Farm. C2 and D2 = cow 157. A4-D4 = positive control.
And was positive again when a second sample was taken.

Another way in which to determine whether #157 was positive for SBV antibodies was to immunolabel some cells infected (or mock infected) with SBV. Serum from #157 clearly detected SBV whereas serum from the other animals didn't react.

(a) the s/p output values from the antibody ELISA of the cows tested following #157's abortion. When serum from #157 was used to immunolabel cells, green signal was only seen in cells infected with SBV.

SBV was present on the farm. At the time, this was several degrees further north than the known distribution. We tested to see whether the antibodies that were recognising the virus were IgM isotype (in which case the infection was recent) or IgG, meaning that the infection was older. Everything was IgG, so the infection had been around for a bit. How long had it been in the area?

In essence this was a 'just in time' scenario; soon virtually all of the UK's farms would be positive for SBV. I found it hard to believe that vet schools weren't already screening their flocks and herds, but here was an opportunity to look at seroprevalence at the herd level in a 'typical' UK commercial dairy farm. So every animal in the herd was sampled and tested for SBV antibodies. An important aspect is that no animals were moved onto the farm during the previous months, therefore the SBV must have arrived by some other means - clearly the likely option being midges. Only a few of the herd were positive, but clearly SBV was present.

During the summer two deformed calves were born. In over 20 years previously, only a single deformity had occurred in this herd. What's more, the dead calves had issues with joints, something that would be consistent with the deformities observed with SBV. One in particular had features that looked extremely similar to those in the literature that had been confirmed as having SBV, including fused and stunted limbs.

A dead calf with clinical features sugestive of SBV, including stunted and fused joints, most obviously a suggestion of arthrogryposis in the hind legs.

The other calf seemed quite the opposite, as if there were no joints, resulting in a floppy carcass, even if the calf otherwise (outwardly) looked fine. 

A deformed calf born in the summer, with a 'bag of bones' type deformity.

Two deformed calves, knowing that SBV had been present at the crucial time, certainly seemed suggestive that these deformities were as a result of SBV. But this isn't in the paper as we can't state that they were the result of SBV without testing them for the presence of the virus. A post mortem would have been revealing.

Another thing that is rather superficial in the paper is another aspect often associated with SBV, changes in milk yields. Overall there was a depression in the milk yield, but there's no way of proving that this was not because of some other factor. What was more dramatic were sudden acute periods of no milk combined with what appeared to be severe depression, but again it's impossible to say that this was as a result of SBV. If we'd tested these animals and it had coincided with SBV viraemia, then perhaps we could say it was related. In reality, these acute episodes are the most commonly observed clinical feature of SBV, at least in cattle, with the deformed offspring representing the exception rather than the norm - there were many other calves born that were perfectly fine. Somewhat frustratingly it is the deformities aspect that people most want associated with SBV, thus that's what dominates in SBV papers and talks, including this paper. It's difficult to nail this kind of thing down though as everything is by association rather than causation, and in many cases is difficult to measure, e.g. how do you know a cow is feeling rough due to SBV? 
In the paper there's a mention of high levels of diahorrea. Again, this is difficult to a) quantify and b) inextricably link to SBV circulation in the herd.

When it got colder, and the midge season was theoretically over, we tested again. This time the majority of the animals were positive for SBV antibodies. Clearly SBV had spread throughout the herd over the summer period.

The proportions of animals in the milking herd that were positive for SBV either before or after the summer period.

This is not in the least surprising. A more dramatic result would have been if there had been any other outcome. The interesting fact though is that, during the summer, the milking cows were at pasture for only a few days. Dogma until a few years ago was that midges are generally reluctant to enter livestock housing. This was based primarily upon observations in the field of Bluetongue virus (BTV), where the Afro-Asiatic species C. imicola is the key vector. It is now established that European midges are perfectly happy to enter buildings. As well as exposure to vectors, another key driver of arbovirus transmission is temperature, which affects both the biting behavior of the vector, and also the kinetics of virus replication within the midges. When the first case in #157 was found, the temperature was only around 10 degrees. The obvious caveat here being that this temperature is the outside temperature; inside it's warmer - that temperature would be very interesting to know. This is relative though: it may be warmer inside but that's relative to 10 degrees; even if it was 5 degrees warmer that's still only 15 degrees. This is still quite cool.

Overall the message would seem to be that not letting the animals spend their days and nights roaming freely at pasture is no barrier to arbovirus transmission. This perhaps shouldn't be surprising. It's warmer, the breeding habitat is textbook for Culicoides, the animals are closer together, there's no wind etc. and there is, inevitably still exposure to the outside.
I'm clearly biased, but the beauty of this study remains that it reflects a real situation. This is not a controlled vet school farm. It is not a sentinel herd kept to check for the first incursion. It is a working dairy farm that more accurately reflects the average scenario for the UK.

And lastly, in case you wondered, #157 has a name: Blossom.

A. E. Shaw, D. J. Mellor, B. V. Purse, P. E. Shaw, B. F. McCorkell, M. Palmarini. (2013). Transmission of Schmallenberg virus in a housed dairy herd in the UK Veterinary Record DOI: 10.1136/vr.101983