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 Brüning-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

Monday, 7 October 2013

Filming fluorescent Marburg virus
For some Marburg is a city in Germany. It's also the name of a virus closely related to the much more widely known Ebola virus (a name which people tend to associate with a virus as opposed to the small river it's named after). What they both have in common, beyond both being members of the Filoviridae family,  is a propensity to induce highly unpleasant, and often lethal, haemorrhgagic fevers. Marburg virus (MARV) first surfaced in 1967 in laboratory workers in Marburg and Yugoslavia and, just like Ebola, has caused sporadic cases and outbreaks since then, the most horrifying of which was in 2004-2005 in Angola, where 227 of 252 (90%) of those known to be infected died.

The worm-like form of Marburg virus particles. 
As per many viruses, much about the MARV lifecycle within the cell remains a mystery. A recent paper in PNAS used live cell imaging to dissect some of the events involved in making new viruses and how they shuttle to a point of release. 

Live cell imaging is often based upon fluorescence, so one of the first things was to make the tools. Essentially, they made versions of structural viral proteins, VP30 and VP40, that are tagged with a fluorescent molecule. To VP30 they added green fluorescent protein (GFP) and showed that when expressed from a plasmid it behaved like the untagged VP30. Similarly, they inserted a red (RFP) version of VP40 into the genome of the virus, such that wild type (wt, = unmodified) and tagged VP40 were produced. The new virus behaved similarly to the unmodified virus, at least early during infection. In infected cells, RFP-VP40 colocalised with wt VP40, implying that this modification didn't alter its localisation. Tools made.

The first step of virus production/release they looked at was the exit of nucleocapsids from the inclusions where nascent viruses are thought to assemble. When they filmed inclusions, VP30-GFP was seen to be leaving, confirming this is where nucleocapsids are assembled, but not with RFP-VP40 (despite VP40 being present at the inclusion body), leading to the conclusion that VP40 is added elsewhere.

Nucelocapsids leaving the inclusion. Individual nucleocapsids could be seen leaving (top) and of those leaving, VP30 but not VP40 was present (bottom) 

If the VP40 component of particles is being added somewhere other than the inclusion, then the obvious question to ask is 'where does VP40 get added?'. Again, they turned to microscopy. When they counted the number of nucleocapsids containing both VP40 and VP30, the number increased towards the plasma membrane, implying that it is here that VP40 associates with the nucleocapsids.

VP40 gets added near the plasma membrane: the closer a nucleocapsid is to the plasma membrane, the greater the likelihood that it also contains VP40, suggesting that it is at the cell periphery that VP40 becomes associated with nucleocapsids.

A  bonus of filming an infection is that there is an additional parameter, i.e. time. This means that the speed at which things happen can be worked out. In this case the authors were able to work out that the nucleocapsids moved at up to 500 nm/sec. On top of that, they were able to figure out that the movement was quicker towards the centre of the cell, as opposed to more remote regions, possibly because nucelocapsids are using different motor proteins in different regions to surf the cytoskeleton. But which component of the cytoskeleton? Two approaches were used. First, they filmed VP30-GFP labelled nucleocapsids in cells with either red tubulin or red actin: only in the case of actin was the movement consistent with riding a particular filament .

In the second approach, treating cells with nocodazole, which disrupts microtubules, had no effect whereas cytochalasin D disruption of the actin filaments brought MARV movement to a halt. In both cases actin appears to be the answer.

Lastly, they looked at the presence of the nucleocapsids in the filopodia extruded from the infected cells. From their observations, they concluded both that VP40 must be associated with the nucleocapsids, and that the motor protein Myosin 10 (Myo10) is involved in the transport of nucleocapsids in the filopodia.

This work is impressive for many reasons. most immediately obvious is the reliance upon live cell imaging. Very often there are the (reasonable) requirements for observations in the microscope to be backed up by biochemical data. It's a great example of what can be achieved via deductions made from observations in careful experiments. The difficulty in doing this work is also easy to overlook. I get the impression that doing this project in BSL-4 would be tricky. Conveniently, they had a remote controlled microscope that they could operate from a more comfortable location, something that's rather handy if you're going to film fluorescent Marburg.

Gordian Schudt, Larissa Kolesnikova, Olga Dolnik, Beate Sodeik, and Stephan Becker (2013). Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances Proceedings of the National Academy of Science DOI: 10.1073/pnas.1307681110

Saturday, 28 September 2013

Bluetongue, Schmallenberg.......Oropouche?
Until relatively recently, Culicoides midges were probably most famed for being an annoyance to people walking, camping, fishing etc. in the Scottish hills. And this is justified - a pub I went to this summer even provided a selection of repellents for their customers. Horse owners most likely associate them with sweet itch, perhaps also that they transmit African Horse Sickness. Many UK and European cattle and sheep farmers would not have thought Cuilcoides were of any significance beyond being a nuisance to their animals.

Next to the whisky and Tennents this Highlands pub provides insect repellents for its customers.

This all changed in 2006, when a severe strain of the Cuicoides transmitted Bluetongue virus (BTV) seemingly parachuted into the middle of Northern Europe and spread wildly, with high levels of morbidity and mortality. The following year BTV arrived into the UK, probably as a result of infected midges being blown across the channel. Fortunately for UK farmers BTV didn't get very far that summer, and by the following year there was a vaccine available. Nevertheless, Culicoides were now very much in the conscience of farmers, which meant that they were fully aware of what may happen when Schmallenberg virus (SBV), another Cuicoides transmitted virus, was discovered in Germany in 2011. Sure enough, a large proportion of the UK sheep and cattle are likely to now be immune to SBV as a result of it sweeping the country in the last couple of years.

The obvious question to ask is, 'what about humans?'. There are several other livestock viruses that are spread by Culicoides, most importantly African Horse Sickness Virus, but if livestock viruses can spread so dramatically, what would happen if a Culicoides-borne virus arrived that could infect humans. The obvious candidate is Oropouche virus, which is from the same family of viruses, the Bunyaviridae, as SBV. This is discussed as an example in a recent paper by Carpenter et al discussing the impact of Culicoides on public health. In the case of OROV, C. paraensis is the primary vector involved in epidemics that occur in south America. An interesting, and potentially important, fact is that the biting patterns of C. paraensis are different from the Culicoides species found in the UK and Northern Europe. Whereas C. paraensis bites at a low rate during both day and night with relatively little impact upon human behaviour, European midges bite so aggressively that people will often seek shelter, effectively reducing exposure levels.

Full-size image (152 K)
Where do midges occur? A) the overlap of livestock and Culicoides,
 and B) the overlap of farmland (i.e. habitats for midges) with urban areas. From Carpenter et al 2013.

If you ignore the aspect of whether there's a virus that fits the requirements, the authors put forward some reasons why an outbreak is perhaps unlikely. Firstly, the biology of the vector may simply mean that not enough survive long enough to transmit an arbovirus. Secondly the habitats of European midges don't tend to overlap with humans as much, thus reducing exposure. And thirdly European midges are seasonal, as opposed to the year-round presence of C. paraensis. All of this suggests that Culucoides may be of relatively limited impact for the transmission of viruses among humans, although they may still facilitate spillover events from animals to humans. For the time being it appears that, where viruses are concerned, Cuicoides appear to be of more importance for livestock diseases. What is for sure though, is that they will remain a pain for anybody wandering the Scottish hills in summer.

Carpenter S, Groschup MH, Garros C, Felippe-Bauer ML, & Purse BV (2013). Culicoides biting midges, arboviruses and public health in Europe. Antiviral research, 100 (1), 102-113 PMID: 23933421

Tuesday, 30 July 2013

Hantavirus and leaky vessels
I once saw a video in which the lung of a Bluetongue Virus (BTV) infected sheep was cut open at post-mortem. As the scalpel cut in, it was clear the lung was full of fluid. Lungs full of fluid don't work very well. As a result, sheep infected by BTV, as well as horses infected with African Horse Sickness Virus, will often die by drowning simply as a result of their vasculature leaking fluid into the lungs. I've often considered this reminiscent of what happens with Hantavirus pulmonary syndrome (HPS), caused by new world hantaviruses, including Andes virus and Sin Nombre virus (the latter of which causes sporadic outbreaks across North America). In Eurasia there are related viruses, including Hantaan virus, which cause Hantavirus haemorrhagic fever with renal syndrome (HFRS). Although different, both diseases involve vascular leakage.
A deer mouse: the wild reservoir of hantaviruses in the New World. 
Endothelial cells, those that line the capillaries and other blood vessels, aren't damaged during infection, so how do the vessels become leaky? One suggestion has been that it occurs as a result of the cytokine arm of the immune response, although removing the T cells modulating cytokines appears to have limited impact upon pathology. An alternative hypothesis is that vascular endothelial growth factor (VEGF), which is reportedly elevated during hantavirus infections, degrades vascular endothelium cadherin (VE-cadherin), a molecule with importance for vessel integrity. A recent paper in PLoS Pathogens by Taylor et al contradicts some of this, and suggests a further possibility: activation of the Kallikrein-kinin system (KKS),which leads to the release of bradykinin (BK). BK in turn is an inflammatory molecule that leads to vasodilation and increased vascular permeability.

Although nothing can replicate the real thing, the authors created their own capillaries in a dish and showed that they could be infected. When they looked for a decrease in VE-cadherin (as per hypothesis 2 mentioned above), the levels appeared more or less equal regardless of infection, likewise the amount of VEGF released from the artificial capillaries did not alter significantly as a result of infection.

Home made capillaries: Hantavirus infection (indicated by the presence of nucleocapsid) appears not to alter the expression of vascular endothelial cadherin. Similarly, capillaries infected with ANDV or HTNV still produce VEGF. 

When they looked for BK release as a result of activating the KSS, they found a dramatic increase when the capillaries, or the cells which are used to make the capillaries, were infected with either Hantaan or Andes virus and treated with molecules that would be found in the blood stream of infected patients (FXII, PK and HK). This implies that hantavirus infection induces permeability as a result of a more active KKS.

BK production by cells infected with hantaviruses: Cells infected with HTNV or ANDV produce more BK (and by extension enhanced permeability) relative to mock infected, although less pronounced in the case of pulmonary artery smooth muscle cells (PaSMC).

Clearly, the KKS was working OK, but more so in cells infected with the viruses. An important aspect is the cleavage of HK which ultimately leads to the release of BK. The authors looked at HK cleavage in the presence of FXII and found that cleavage was enhanced in its presence. Going further, when they added an inhibitor of FXIIa (the activated form of FXII), they found the cleavage no longer occurred; FXII is clearly therefore required in the system. 

Measuring resistance: resistance is used as an indication of permeability. Infected cells have enhanced permeability when the KKS factors are applied (A). B-D show the effect on mock, HTNV or ANDV infected cells with the inhibitors CTI (blue) PKSI-527 (red) or HOE 140. 
All of these, and some other, experiments suggest that the KKS and BK liberation may, at least in part, be responsible for the leaky vasculature as a result of hantavirus infection. To look at this the authors used electric cell-substrate impedance sensing to measure the resistance/permeability (leakiness) of confluent layers of endothelial cells infected (or not) with hantaviruses. Cells that were infected, i.e. effectively had an activated KKS and BK present, showed a decrease in cell resistance of up to 50%, compared to a maximum drop of only 10% observed in uninfected cells. The addition of inhibitors against the KSS altered the pattern, reducing the effect observed in infected cells, thus directly implicating alterations in the KKS as being the cause of these changes.

The KKS system and Hantavirus infection: HK and PK are enhanced on the surface of infected cells, leading to cleavage of HK (involving FXII) and subsequent release of BK, and thus enhanced permeability of the endothelial layer. Several drugs are available which target the pathway.

It remains possible, indeed likely, that there are other factors that control the vascular permeability and therefore pathogenesis of hantaviruses. Lungs filling with fluid isn't unique to hantaviruses and there are likely several mechanisms yet to be deciphered. This study does though highlight a new pathway of interest that leads to leaky vessels, and, importantly a pathway for which there are inhibitors: perhaps the leaks can be mended.

Taylor, S.L., Wahl-Jensen, V., Copeland, A.M., Jahrling, P.B., Schmaljohn, C.S. (2013). Endothelial Cell Permeability during Hantavirus Infection Involves Factor XII-Dependent Increased Activation of the Kallikrein-Kinin System PLoS Pathogens DOI: 10.1371/journal.ppat.1003470

Friday, 5 July 2013

What's Killing the Koalas?
My current employer made his name working on a retrovirus: Jaagsiekte sheep retrovirus (JSRV). JSRV is a betaretrovirus whose greatest claim to fame is killing Dolly the sheep, but it has revealled many aspects of sheep and retrovirus biology. One of the attributes most associated with viruses is that they're obligate intracellular parasites: without a cell to replicate in, viruses are often little more than a bunch of molecules. Retroviruses take this to the extreme and insert into a cells genomic DNA in order to replicate. At it's simplest this involves the virus like any other infecting a somatic cell, intergrating into the cellular DNA, replicating and exiting. This is the 'exogenous' form. 

Alternatively, if the cell happens to be a germ cell, from which sperm or eggs are produced as a precursor to another individual, the retroviral DNA will be inherited by Mendelian inheritance as for any other gene. When this happens, the virus is now regarded as being 'endogenous'.

In the last few years a type-C retrovirus, Koala retrovirus (KoRV), has been linked to the development of Koala immunodeficiency syndrome (KIDS). As the term suggests, KIDS results in a depleted immune system, resulting in enhanced vulnerability to infectious diseases and cancers. KIDS has become a prominent killer of koalas, particularly those in captivity, where the majority of studies have been performed. KoRV's closest relative appears to be gibbon ape leukemia virus (GALV) which, like KoRV, causes lymphomas and leukemia.

KoRV represents an example of a very young endogenisation event, with the intergation event thought to be only around 100 years ago, and the integrated copies are able to generate infectious viruses. A recent paper in PNAS describes the isolation of a variant of KoRV in San Diego and Los Angeles zoos (Xu et al 2013). All of the koalas at the zoos contained the endogenised form of KoRV. However, in six koalas at Los Angeles zoo, including 3 that died, they found an additional, slightly different, KoRV sequence (KoRV-B, as opposed to the original KoRV-A). The majority of the changes were in the U3 region of the long terminal repeats (LTR). Particularly striking was that parts of the virus responsible for binding to the cell receptor looked more like those of an exogenous virus as opposed to the more endogenous form possessed by KoRV-A. Indeed, when they looked at receptor usage, KoRV-B used a different receptor to KoRV-A and GALV.

Mother to Joey transmission: Joeys only become infected with KoRV-B when the dam is infected, even if the sire is positive, implying the virus is transmissible rather than inherited. (Xu et al 2013)

Further evidence of the infectious nature of KoRV-B came from the observation of infected (or not) joeys born to infected (or not) parents in a family at Los Angeles zoo. A positive joey was only born when the mother was positive; it was possible for a joey to be born negative for KoRV-B even if the father was positive for KoRV-B (as long as the mother is negative).

Koalas would appear in a bad way. However, endogenous retroviruses aren't necessarily harmful. On the contrary, some may be beneficial. A prime example is the JSRVs. The presence of endogenous JSRVs results in so-called 'late restriction'. Interference of exogenous JSRV replication by the presence of endogenous JSRVs is ultimately beneficial for the sheep.

Endogenous JSRVs: a variety of genomic arrangements of JSRVs found in the sheep genome. (Arnaud et al 2007)

Inevitably, as the sheep genome is targeted in further rounds of infection by exogenous JSRVs, a tug of war develops such that a balance exits between the late restriction imparted by endogenous JSRV(s) and the exogenous JSRV. The sheep genome has been invaded multiple times by JSRV, to the extent that the domestication of sheep can be traced based upon which endogenous JSRVs are present in the genome of a particular sheep breed (Chessa et al 2009).
Whether it's too late for something similar with the koalas, time will tell.

Wenqin Xu, Cynthia K. Stadler, Kristen Gorman, Nathaniel Jensen, David Kim, HaoQiang Zheng, Shaohua Tang,, & William M. Switzer, Geoffrey W. Pye, Maribeth V. Eiden (2013). An exogenous retrovirus isolated from koalas with malignant neoplasias in a US zoo Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1304704110

Arnaud F, Caporale M, Varela M, Biek R, Chessa B, Alberti A, Golder M, Mura M, Zhang YP, Yu L, Pereira F, Demartini JC, Leymaster K, Spencer TE, & Palmarini M (2007). A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses. PLoS pathogens, 3 (11) PMID: 17997604

Chessa B, Pereira F, Arnaud F, Amorim A, Goyache F, Mainland I, Kao RR, Pemberton JM, Beraldi D, Stear MJ, Alberti A, Pittau M, Iannuzzi L, Banabazi MH, Kazwala RR, Zhang YP, Arranz JJ, Ali BA, Wang Z, Uzun M, Dione MM, Olsaker I, Holm LE, Saarma U, Ahmad S, Marzanov N, Eythorsdottir E, Holland MJ, Ajmone-Marsan P, Bruford MW, Kantanen J, Spencer TE, & Palmarini M (2009). Revealing the history of sheep domestication using retrovirus integrations. Science (New York, N.Y.), 324 (5926), 532-6 PMID: 19390051

Sunday, 23 June 2013

Agricultural Shows; a virus' dream?

This week saw the annual event when farmers descend from all parts of Scotland to compare their animals: The Royal Highland Show. I went along and, after a while wandering around the tractors, had a look around the livestock. Looking at the plethora of pampered sheep and cattle, a few things occurred to me. 
First was a half-hearted feeling of 'missed opportunity'. Sheep in particular, but also cattle, had come from across Scotland (and beyond). Imagine if a blood sample of every animal had been taken. Schmallenberg virus (SBV) is known to have reached, and circulated, in Scotland, but nobody knows to what extent. Perhaps testing these animals may have given a cross-section of SBV distribution across Scotland. Maybe even an idea of variation in breed susceptibility; although there's nothing really suggesting that such differences exist.

Show time: a Jacob sheep gets a final trim in the sheep yard prior to showing at the Royal Highland Show.
Second was considering the potential for transmission and dissemination of pathogens across wide geographical areas. In 2001 in the UK the dispersal of Foot and Mouth Disease Virus to various regions was due, at least in part, to the mixing and distribution of sheep from Hexham Market. Agricultural shows represent a similar type of mixing event, and last for days rather than hours. In the case of Foot and Mouth, the clinical signs are mild in sheep, thus allowing infected animals to go unnoticed.

Bringing animals to the show with clinical signs of disease is unlikely to happen as the idea is obviously to show the best animals. That restricts the possibility of infectious animals to those in a pre-clinical incubation stage. However, shows lasting for several days gives sufficient time in which to develop a viraemia and allow transmission, either via direct contact, fomites or vectors. Unsurprisingly there are already studies on the role of agricultural shows. One regarding UK shows (Webb, 2006) revealed competitors at shows to form a large network, with some competitors travelling to shows up to 600 km apart. Clearly there is potential, but thus far nothing seems to have been nailed down showing an outbreak resulting from mixing at agricultural shows.

Fig. 4
The impact of time-between shows: the number of days between shows affects the network, no time limit results in a mass network (a) whereas time limits of 14 days (b), 10 days (c) or 7 days (d) reduce the number of nodes, effectively separating shows and breaking links. From Webb 2006.

Third was the zoonotic potential.
It is well established that  cross-species transmission of zoonotic pathogens occurs at the interface between the animal source and humans. These shows represent perfect scenarios for such interactions. Largely absent from the Royal Highland Show were pigs, which have previously been shown to be a source of influenza A infection. Pigs may therefore represent more of a risk due to their ability to sustain various zoonotic agents. Likewise shows where chickens and ducks are present, offering the possibility for the transmission of avian influenza.  

Odd choice: A sheep decides a coat is preferable to hay.

The idea of catching bugs from farm animals at these kind of events, as well as at petting farms for kids etc., is not new. And it is true, infections do happen. The knee-jerk response of a modern nanny state society, at least in the UK, would therefore dictate the banning of such scenarios, including all agricultural shows without question. But this is another case of putting risk into perspective; of all the millions of animals, and the millions of contacts with humans that have happened at these shows over the years, how many infections have occurred, or at least been serious enough to cause alarm? The simple answer: not many. For now though, the relative importance that agricultural shows play in the transmission, evolution and species transfer of viruses remains largely unknown.

Webb, C.R. (2006) Investigating the potential spread of infectious diseases of sheep via agricultural shows in Great Britain. Epidemiol Infect. 134(1)31-40. doi: 10.1017/S095026880500467X

Sunday, 9 June 2013

Françoise Barré-Sinoussi visits the Centre for Virus Research
Few stories in virology can have been more frequently or comprehensively covered as the early days of the HIV/AIDS epidemic. Randy Shilts' 'And the Band Played On' is surely one of the most detailed accounts and, whilst massive, provides insights into what went on in the first few years of the epidemic. If you don't fancy reading the book, there's always the resulting film to watch, although it concentrates much more on the heroics of the epidemiologist Don Francis. 

And The Band Played On: Don Francis fights HIV in the face of politics and business.
There are so many aspects discussed both in the book/film and in general about the discovery of HIV as the cause of AIDS, but for me it is the politics which strikes the hardest. The political scene was set for the emergence of a disease like AIDS; a disease emerging largely in sections of society which a conservative government would rather not acknowledge. The original CDC Morbidity and Mortality Weekly Report  MMWR) is rather poignant, now that we know what it represented - I've pasted it at the end (copied from the CDC Website). 

Scientifically, politics became an issue too. A rather bitter rivalry built up between two particular virology groups as everyone raced to figure out the ultimate cause of AIDS. Now it seems obvious that it was a virus attacking T lymphocytes, but at the time things were much more unknown. As an example, one red herring in the early days was poppers/alkyl nitrates as a result of their association with homosexual bathhouses. Ultimately the rivalry ended in dispute as to who had discovered the viral cause of AIDS - something that, in theory, should have taken second place over concentrating on doing something about it. In the end, the French group lead by Luc Montagnier, (as opposed to the US group lead by Robert Gallo) are generally regarded as being the discovers of HIV - the cause of AIDS - and Montagnier won the Nobel Prize in Physiology or Medicine in 2008. Together in receiving the prize was Françoise Barré-Sinoussi. In 1983 Barré-Sinoussi published a paper in Science reporting the isolation of a retrovirus from a suspected patient - effectively reporting the viral cause of AIDS.

The first step was isolating the virus. Cells from a lymph node biopsy from the patient were cultured in growth medium. Samples were taken over several days, and tested for reverse transcriptase (RT) activity (a hallmark of retroviruses). After 15 days they detected RT activity, suggesting a retrovirus was present. When some of the 'infected' cells were mixed with healthy cells, RT activity was again detected, but only after 15 days, therefore suggesting that transmission had occurred. Media without the cells could also infect umbilical cord lymphocytes, and when these were looked at using electron microscopy, viruses could be seen budding from the cells.

HIV budding from the cell membrane

Importantly, Barré-Sinoussi showed that, in addition to isolating a virus with a predilection for human T cells, it was different to the known human T cell leukaemia viruses (HTLV) discovered to that date (largely based upon serum cross-reactivity, but also the fact that this new virus killed the T cells, as opposed to causing cancer in the case of HTLV-I and -II), backing up data that the genetic sequence was distinct from HTLV-I and -II . Altogether the data pointed towards a retrovirus as being behind AIDS. 

Françoise Barré-Sinoussi receives the Nobel Prize for Physiology or Medicine in  2008.

Gallo was at times vilified, some might argue rightly so, but equally his work on T cells and viruses was fundamental towards the ability to isolate the virus in the first place. Thankfully things have placated somewhat and, when he visited the CVR, he gave an interesting talk about his views on what it is going to take to make a good HIV vaccine. My expectation is that Françoise Barré-Sinoussi's story will be equally enthralling.

The following MMRW essentially represents the first report of HIV:

Pneumocystis Pneumonia -- Los Angeles

MMWR 1981;30:250-2 (June 5, 1981)
In the period October 1980-May 1981, 5 young men, all active homosexuals, were treated for biopsy-confirmed Pneumocystis carinii pneumonia at 3 different hospitals in Los Angeles, California. Two of the patients died. All 5 patients had laboratory-confirmed previous or current cytomegalovirus (CMV) infection and candidal mucosal infection. Case reports of these patients follow.

Patient 1: A previously healthy 33-year-old man developed P. carinii pneumonia and oral mucosal candidiasis in March 1981 after a 2-month history of fever associated with elevated liver enzymes, leukopenia, and CMV viruria. The serum complement-fixation CMV titer in October 1980 was 256; in May 1981 it was 32.* The patient's condition deteriorated despite courses of treatment with trimethoprim-sulfamethoxazole (TMP/SMX), pentamidine, and acyclovir. He died May 3, and postmortem examination showed residual P. carinii and CMV pneumonia, but no evidence of neoplasia.

Patient 2: A previously healthy 30-year-old man developed P. carinii pneumonia in April 1981 after 5-month history of fever each day and of elevated liver-function tests, CMV viruria, and documented seroconversion to CMV, i.e., an acute-phase titer of 16 and a convalescent-phase titer of 28* in anticomplement immunofluorescence tests. Other features of his illness included leukopenia and mucosal candidiasis. His pneumonia responded to a course of intravenous TMP/SMX, but, as of the latest reports, he continues to have a fever each day.

Patient 3: A 30-year-old man was well until January 1981 when he developed esophageal and oral candidiasis that responded to Amphotericin B treatment. He was hospitalized in February 1981 for P. carinii pneumonia that responded to oral TMP/SMX. His esophageal candidiasis recurred after the pneumonia was diagnosed, and he was again given Amphotericin B. The CMV complement-fixation titer in March 1981 was 8. Material from an esophageal biopsy was positive for CMV.

Patient 4: A 29-year-old man developed P. carinii pneumonia in February 1981. He had had Hodgkins disease 3 years earlier, but had been successfully treated with radiation therapy alone. He did not improve after being given intravenous TMP/SMX and corticosteroids and died in March. Postmortem examination showed no evidence of Hodgkins disease, but P. carinii and CMV were found in lung tissue.

Patient 5: A previously healthy 36-year-old man with a clinically diagnosed CMV infection in September 1980 was seen in April 1981 because of a 4-month history of fever, dyspnea, and cough. On admission he was found to have P. carinii pneumonia, oral candidiasis, and CMV retinitis. A complement-fixation CMV titer in April 1981 was 128. The patient has been treated with 2 short courses of TMP/SMX that have been limited because of a sulfa-induced neutropenia. He is being treated for candidiasis with topical nystatin.
The diagnosis of Pneumocystis pneumonia was confirmed for all 5 patients ante-mortem by closed or open lung biopsy. The patients did not know each other and had no known common contacts or knowledge of sexual partners who had had similar illnesses. The 5 did not have comparable histories of sexually transmitted disease. Four had serologic evidence of past hepatitis B infection but had no evidence of current hepatitis B surface antigen. Two of the 5 reported having frequent homosexual contacts with various partners. All 5 reported using inhalant drugs, and 1 reported parenteral drug abuse. Three patients had profoundly depressed in vitro proliferative responses to mitogens and antigens. Lymphocyte studies were not performed on the other 2 patients.

Reported by MS Gottlieb, MD, HM Schanker, MD, PT Fan, MD, A Saxon, MD, JD Weisman, DO, Div of Clinical Immunology-Allergy, Dept of Medicine, UCLA School of Medicine; I Pozalski, MD, Cedars-Mt. Sinai Hospital, Los Angeles; Field Services Div, Epidemiology Program Office, CDC.

Barre-Sinoussi, F., Chermann, J., Rey, F., Nugeyre, M., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W., & Montagnier, L. (1983). Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS) Science, 220 (4599), 868-871 DOI: 10.1126/science.6189183

Thursday, 30 May 2013

Tick-borne encephalitis milk
Based upon the name, Tick-borne encephalitis virus (TBEV) would be expected to be transmitted by ticks. And it appears it is. But it can also be spread by milk/milk products. A recent paper has reported a case in Slovenia where 3/4 people who drank the milk of a goat (later found to be positive for TBEV) became ill. TBEV is a flavivirus and is present in many parts of Europe, with incidence levels in recent years of up to 18.6 cases/100,000 people.
Unpasteurised cheese - a potential source of TBEV infection

Transmission of TBEV in unpasteurised milk hasn't previously been reported in Slovenia specifically, but it was already known that TBEV can be transmitted in raw dairy products. In a recent study, four people had drank unpasteurised colostrum from a goat, three of whom subsequently became sick, having symptoms of fever, malaise, headache, nausea and tremor. Although TBEV RNA couldn't be detected in the blood of any of the three patients, they all had antibodies. IgG and IgM isotypes were detected by ELISA, and the serum was neutralising for the virus. The fourth person, who didn't become ill, had been vaccinated against TBEV and had good antibody titres against TBEV, although IgG titres taken at different intervals suggested they'd received a boost, presumably as a result of drinking the infected milk.

Goats, not sheep seem to be the culprits when it comes to harbouring TBEV in their milk
The authors took samples from the 9 goats and 9 sheep on the farm, and tested them for evidence of TBEV.
Perhaps unsurprisingly, 5/9 goat sera  and 1/4 goat milk samples were positive for TBEV antibodies. The suspect goat whose milk had been drunk prior to the patients becoming sick was also shown to have RNA (indicative of virus) in both the blood and milk, confirming that the goat had evidence of actual viral antigen.

None of the sheep samples were positive, which I find one of the most intriguing points - do ticks prefer goats, or is there species specificity when it comes to TBEV?

According to the authors, drinking unpasteurised milk is fashionable as part of a 'natural lifestyle'. Based upon this paper, the risk of contracting TBEV (as well as other pathogens) would therefore also be part of the natural lifestyle; that's an interesting choice of lifestyle.

Hudopisk, N., Korva, M., Janet, E., Simetinger, M., Grgič-Vitek, M., Gubenšek, J., Natek, V., Kraigher, A., Strle, F., & Avšič-Županc, T. (2013). Tick-borne Encephalitis Associated with Consumption of Raw Goat Milk, Slovenia, 2012 Emerging Infectious Diseases, 19 (5) DOI: 10.3201/eid1905.121442

Friday, 24 May 2013

Schmallenberg virus...where are we at: Part 3. Vaccine time

As Bluetongue virus 8 was making its way to the UK in 2007 I was asked by a farmer whether or not he should vaccinate when the vaccine became available. 100% yes was the answer, and would be now. BTV-8 was a particularly virulent strain, resulting in mass death in sheep and even (unusually for BTV) causing disease and deaths in cattle. Bluetongue had never before been to the UK, so the entire herd was susceptible to the virus. The result was that, whilst East Anglia experienced a few outbreaks in 2007, the vaccine prevented recrudescence of the virus in 2008; unlike what happened on the continent after 2007, where BTV overwintered from 2006 and erupted in 2007 to cause the largest outbreak of BTV in history.

Bluetongue virus did eventually reach the UK, but vaccination stopped it from progressing

This week saw the news that a vaccine for Schmallenberg virus will be available from Merck by the summer. The obvious assumption is that every farmer will be desperate to vaccinate. That would be no surprise considering the damage it's done this past year, in particular to the sheep industry where countless lambs have been lost as a result of the disease.

But the same question now arises - should you vaccinate? This time the situation is very different. We've had it. Would vaccinating now be the equivalent of the metaphorical slamming the stable door after the horse has bolted etc.? If current reports from the continent equate to the UK, then the majority of cows and sheep will be immune, in which case a vaccine might be of limited value. There will however be some naive animals which somehow avoided becoming infected, and once maternal antibody has waned this years offspring will be open to infection.So vaccinate, right?

In the case of human diseases, the choice to vaccinate is generally simple; vaccinate. Similarly, most horse  and pet owners would probably also vaccinate their animals in the face of an emerging disease (assuming a vaccine were available). Farming is different though; farming is to make money. The option to vaccinate is no longer based upon sentimentality, but business. How much do you stand to lose if there's an outbreak, relative to how much it costs to vaccinate when there's only the possibility of an outbreak? In the case of BTV the evidence from the continent was that it would be devastating. The choice to vaccinate was clear.

With SBV however, does it make sense from a business perspective to spend a lot of money to vaccinate the flock/herd? Say 90% of the animals are immune - a reasonable guess based upon studies on the continent. To begin with this level of seroprevalence already makes it more difficult for an outbreak to persist. Ignoring that, and assuming the extent of infection will be the same as last year, 9% of the flock would in theory become infected. If in turn we assume only 10-20% of those infected would be affected clinically, that's only 1-2% of the overall flock/herd.

You're fired: does it make business sense to vaccinate animals when there might not be any need?
In a flock of 1000 animals, 10 animals may be affected. Is that a sufficient number to warrant vaccinating 1000 sheep? Would you vaccinate a herd of 500 cattle on the basis there might be 5 cases of acute disease? Clearly such numbers are approximate, and may be wide of reality, but they're realistic enough to shows there's a difficult choice to make. I can't advise on whether or not to vaccinate, but I do know it's going to be a tricky choice for those involved.

Friday, 17 May 2013

poly(A) messages; lost in translation
From a virus' perspective, how do you translate your own messenger RNA (mRNA), whilst not allowing your host cell to continue manufacturing its own proteins, including those that might be detrimental to virus survival? It's a problem viruses have found various ways to overcome, often by manipulating the biology of the mRNAs, which have the following structure:

The classical polyadenylated mRNA ready for translation

Simply, an eIF4F cap-binding complex binds to the cap and a poly(A) binding protein (PABP) interacts with the poly(A) tail. The PABP in turn interacts with eIF4G of the cap binding complex, thus circularising the mRNA for  efficient translation to occur.

The translation complex showing circularisation enabled by PABP linking the poly(A) tail to eIF4G.

A good way in which to specifically translate viral messenger RNAs is to  make the viral mRNAs different in such a way that viral mRNAs are the most efficiently translated mRNAs. Picornaviruses (e.g. polio, foot and mouth disease) and flaviviruses (e.g. West Nile, Hepatitis C), genomes contain an internal ribosome entry site (IRES), which allows ribosome attachment and subsequent translation in the absence of the 5' cap; get rid of the ability of the cell to translate capped mRNAs and suddenly the viral mRNAs are preferentially translated.

Rotaviruses target the other end. Rotavirus mRNAs all end in a specific sequence ....GACC instead of a poly(A) tail. One of the viral non-structural proteins, NSP3, has been shown to act in similar fashion to PABP; NSP3 specifically binds RNAs ending in this sequence (i.e. rotaviral mRNAs), and also binds the cap-binding complex in place of PABP, but with higher affinity than PABP. The overall result is that polyadenylated mRNAs are outcompeted by rotaviral mRNAs. NSP3 also seems to be responsible for PABP accumulating in the nucleus, where it is unable to translate cytoplasmic mRNAs. Even so, there is evidence that rotaviral mRNA translation appears to be independent of NSP3.

A paper has just come out looking into the location of poly(A), i.e. cellular, mRNAs in rotavirus infected cells.
The first question was, where do you find poly(A) mRNAs in infected cells? Using fluorescence in situ hybridisation (FISH) the authors found poly(A) containing mRNAs to accumulate in the nucleus of cells, thus preventing their translation. Removing NSP3 using RNA silencing prevented this from happening, so that poly(A) mRNAs were then found in the cytoplasm, just as in uninfected cells.

Rotavirus (and NSP3) retain polyadenylated mRNA in the nucleus. Oligo (dT) probes detects polyadenylated (i.e. cell-like) mRNA. Normally the mRNA is distributed throughout the cell (top) in infected cells, signal is only observed in the nucleus (middle row) whereas when NSP3 is silenced, the nuclear block is apparently released (bottom).

Next there is an intriguing finding that, perhaps surprisingly, the untranslated regions (UTRs) of rotaviral mRNAs do not influence how well that particular transcript is translated, as luciferase reporter RNAs with host-cell UTRs were not translated any less efficiently than reporter RNAs with rotaviral UTRs. Most strikingly though, was the fact that the overall efficiency of translation appeared to be enhanced in infected cells, implying that the translation machinery is altered upon infection. These mRNAs were directly transfected into the cytoplasm, which isn't how cellular mRNAs originate. To look at this, the authors used two ways of supplying mRNA to the translation machinery, either directly into the cytoplasm, where they again found it to be more efficiently translated in infected cells, or allowed the cell to transcribe it from a plasmid, in which case they observed a decrease in expression as as result of rotavirus infection. Silencing of NSP3 released this apparent inhibition, with the infected cells appearing more like uninfected cells. Together this all leads to the conclusion that, rather than NSP3 affecting the translation of mRNAs directly, the inhibition of poly(A)-dependent translation is due to a lack of export of newly transcribed RNA.

RNA translation in the presence of Rotavirus: (A) when RNA is  transcribed in the nucleus (and needs to be exported for translation), infection suppresses translation, whereas mRNA transfected into cytoplasm implies that rotavirus infection enhances the translation efficiency of the translational machinery. (B) Suppression of rotavirus NSP3 by RNA silencing removes the block imposed upon protein expression from a plasmid.

The authors checked the location of cellular mRNAs too, and found that they too accumulated in the nucleus of infected cells, whereas in mock-infected cells the mRNAs were found in the cytoplasm. Again, when NSP3 was silenced, this block disappeared.
What happens to the polyadenylated mRNAs which accumulate in the nucleus (alongside the normally cytoplasmic PABP which is also retained in the nucleus)? By looking at the length of the RNAs, and using oligos which target the poly(A) tail, they found that the poly(A) tails were increased in length; an observation in line with data showing that PABP accumulation in the nucleus results in hyperadenylation and nuclear retention of RNAs.
Finally, the authors looked to see whether there were more cellular mRNAs in the nucleus compared to the cytoplasm in infected cells. They found that the cytoplasm of infected cells contained 50% less polyadenylated mRNAs. All of this leads to a scenario in which a translationally very active cytoplasm is (comparatively) free of cellular, polyadenylated mRNAs, into which the virus transcribes masses of its own mRNAs; essentially the viral mRNAs now have the cell's translational machinery to themselves, and all of this apparently orchestrated by NSP3.

As a strategy it makes sense; simply get rid of the host's RNAs.

 Rubio, R., Mora, S., Romero, P., Arias, C., & Lopez, S. (2013). Rotavirus Prevents the Expression of Host Responses by Blocking the Nucleocytoplasmic Transport of Polyadenylated mRNAs Journal of Virology, 87 (11), 6336-6345 DOI: 10.1128/​JVI.00361-13
Piron, M. (1998). Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F The EMBO Journal, 17 (19), 5811-5821 DOI: 10.1093/emboj/17.19.5811