Dengue in Viet Nam

In some, maybe the majority of cases, economic development tends to improve a country's situation regarding infectious disease; a proper sewerage system, for example, may decrease the incidence of diseases associated with the contamination of water sources. Where dengue virus is concerned this is not the case. Dengue is a human virus spread by mosquitoes of the genus Aedes (in particular A. aegypti), which happily breed in dirty water. Economic development tends to result in increased urbanisation and, as a result, ideal breeding conditions are generated for the mosquitoes (tin cans, old tyres etc.). Together, the result is a dense population of humans in the same location as the mosquitoes: in the absence of a vaccine or antivirals dengue has thus thrived. Whilst the conditions are favourable for dengue in general, there are inevitably more specific drivers of transmission and outbreaks.

A recently published study in PLOS Neglected Tropical Diseaes by Rabaa et al looked into what the drivers are for dengue in Viet Nam. The situation in Viet Nam can broadly be regarded as the south (tropical) region being endemic, whilst the north (sub-tropical) is not endemic, but experiences frequent introductions. In central Viet Nam the virus can persist for more extended periods of time, perhaps due to more favourable conditions for transmission and, ultimately, a higher level of immunity. As an illustration as to the impact of urbanisation, Ho Chi Minh city in the south is highly endemic and represents a large source of viruses for the rest of Viet Nam.

The authors compared the dengue serotype 1 (DENV-1) sequences of the envelope (E) gene.

Using a maximum likelihood approach to get an additional grasp of geographical relationships, they found that, perhaps unsurprisingly, all of the sequences belonged to the Southeast Asia subtype of Genotype I.

Phylogeography of DENV-1 genotype 1 in Southeast Asia, 1998-2009. A) Map of north (red) central (yellow) and south (blue) Viet Nam. The colours match in the graphs of mean min/max temperature (B, top graph) and mean precipitation (B, bottom graph), and branches in the maximum clade credibility phylogenetic tree (C). Purple branches represent Singapore sequences.

On the whole, DENV-1 seems to invade subtropical northern Viet Nam regularly, but never seems to become endemic - most likely due to the cold winter temperatures resulting in conditions that are refractory to continued transmission. Such invasions also occur in the central regions, although these persist for longer.

Interestingly, although (as may be expected) within a particular region the diversity among viruses was limited,  on a broader scale Ho Chi Minh City in the south was found to act as a source of virus throughout Viet Nam.

On the other hand, despite local diversity being low, it's interesting that geographically long distance movements were observed in a time-scale that precludes the hypothesis that it's merely natural spread via vectors. Instead, it appears that the movement of infected humans is responsible for seeding at least some of the regions. This is one route by which the north can be seeded. However, because the north is sub-tropical, there comes a time in the year when the vectors die off and transmission is reduced; a familiar scenario with non-tropical arboviruses. 

As interesting a piece of work as this is in itself, it arguably demonstrates something important at a more global level. Clearly DENV can be seeded in different regions by people moving around Viet Nam; if this can happen within Viet Nam, then it's not a massive step to extend Viet Nam to the world.

Maia A. Rabaa, Cameron P. Simmons, Annette Fox, Mai Quynh Le, Thuy Thi Thu Nguyen, Hai Yen Le, Robert V. Gibbons, Xuyen Thanh Nguyen, Edward C. Holmes, John G. Aaskov (2013). Dengue Virus in Sub-tropical Northern and Central Viet Nam: Population Immunity and Climate Shape Patterns of Viral Invasion and Maintenance PLOS Neglected Tropical Diseases DOI: 10.1371/journal.pntd.0002581

Bats vs. Rats

If I had to name one book that got me interested in viruses it would be 'Virus X: understanding the real threat of the new pandemic plagues', by Frank Ryan. The book largely concentrates upon virus emergence; why it happens, where the viruses come from, and what that might mean for the future. Whether or not I now agree fully with everything that's hypothesised is a different matter, although an honest evaluation is difficult considering the advances in science since its publication (1997). Nevertheless, it got me interested at the time.

Flicking back through it last night I read a line regarding virus reservoirs that stood out. "The threat to humanity derives in particular from rodents". This was the logical conclusion derived from the fact that rodents are the most numerous mammal, which is fair enough. Nowadays it's almost all about bats. In the book Ryan does point out the suspicions of bats as reservoirs, but overall in this book the potential significance of bats is over shadowed by a focus on rodents.

With our current knowledge of virus natural reservoirs (a term itself worthy of debate), a suggestion that anything other than bats are the most important source of viruses as far as public health is concerned, is likely to be met with an element of scorn. Bats do indeed harbor a lot of viruses, as was published earlier this year. In this particular paper the authors also estimated the numbers of viruses still to be discovered (320,000), although wisely they also stated that such a calculation was based upon some rather large assumptions:

"Several important limitations must be considered in our extrapolations, including (i) the assumption that a mean of 58 viruses per species is a reasonable estimate and that host populations are panmictic with respect to viral transmission (such that expanded geographic sampling would not influence viral detections), (ii) the assumption that viruses are not shared by more than one host species, (iii) that only those viruses within the nine families are considered in this estimation, (iv) that the results are limited by the sensitivity and specificity of our tests, and (v) that a similar mean cost of sample collection is incurred across all species."  (Anthony et al. 2013, mBio) 

Nevertheless, it's a useful number to have.
Bats have long been suspected as reservoirs, and in the case of rabies it had been firmly established, but I'm not sure when exactly they became so popular for virus hunters. Perhaps around the time of Nipah and Hendra emergence. Nowadays everybody seems to be hunting for viruses in bats specifically.

Bats, it can safely be said, represent an important source of novel as well as known viruses. In terms of virus emergence and spread however, there is more to it. Yes, bats may harbor a lot of viruses. And perhaps yes for one reason or another those viruses may have a higher chance of being unpleasant. But there is more to epidemiology than simply the source. Spillovers in a forest/rural setting are inevitable, and in this case bats pose as much of a risk as rodents. However, the majority of people live in urban areas. And from this perspective, rodents are surely of greater importance for transmission as their populations are so intimately linked with humans. More contact means a greater likelihood of transmission. One of the worst epidemics in history, the Black Death (admittedly caused by a bacterium), was closely linked with rats. More recent, viral, examples include the Sin Nombre hantavirus in New Mexico, and the Arenaviruses (e.g. Lassa fever virus).

It could be argued that, because we've had so much interaction with rats over the years we're unlikely to find anything new. That doesn't mean they're of lesser importance; Lassa fever and Sin Nombre are responsible for the death of more people than those caused by more exotic viruses such as Ebola virus.

Global air travel: 'emergence hotspots' such as South East Asia experience more international travel than central Africa. Image: Max Planck Institute for Dynamics and Self-Organisation/ Dirk Brockmann http://www.ds.mpg.de/3004/news_publication_4406928?c=148862

It is clear that the jungles and savannas of Central Africa harbor bats with viruses of great danger to humans. But is this more important than, say, the populations of rats in densely populated urban centers in South East Asia? Human traffic to and from such urban areas is higher, enhancing the probability of an infection spreading to other parts of the world; would SARS, for example, have spread so far if it had emerged in Uganda? In the future it may be that the world is equally connected. For now though, some places remain more connected than others, and this should be remembered when people decide what's more important: bats or rats.

Anthony SJ, Epstein JH, Murray KA, Navarrete-Macias I, Zambrana-Torrelio CM, Solovyov A, Ojeda-Flores R, Arrigo NC, Islam A, Ali Khan S, Hosseini P, Bogich TL, Olival KJ, Sanchez-Leon MD, Karesh WB, Goldstein T, Luby SP, Morse SS, Mazet JA, Daszak P, & Lipkin WI (2013). A strategy to estimate unknown viral diversity in mammals. mBio, 4 (5) PMID: 24003179

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

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

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

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.

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

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