Wednesday, 19 December 2012

The end of Koch's postulates
How do you know whether disease X is the result of infection by pathogen Y? 
In 1890 Robert Koch published a list of 'postulates' which would form the foundation of ascribing disease causation from that day - until now. Though Koch established his list based upon his studies of bacteria, more specifically Mycobacterium tuberculosis and Bacillus anthracis, (the causative agents of tuberculosis and anthrax respectively), his postulates could be applied more widely. No more would infectious diseases be merely regarded as mysterious happenings. Scientists could now pursue and nail down the cause of a disease by fulfilling Koch's postulates. There are many ways of expressing the same thing, but Wikipaedia describes them as:

1) The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms. 
2) The microorganism must be isolated from a diseased organism and grown in pure culture.
3) The cultured microorganism should cause disease when introduced into a healthy organism.
4) The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Association vs. causation will always be a key issue when it comes to diagnosis and Koch's postulates, as important as they are, soon start to fall apart under closer scrutiny. For instance, what happens in circumstances where the organism can't be isolated in culture - a not unusual occurrence in the world of virology? 

The most significant problems occur though simply due to words such as 'must'. Infectious disease is never so clear cut. Koch himself acknowledged as much during his studies of cholera; whilst the causative agent, Vibrio cholerae, could be isolated from people with cholera, it could also be isolated from healthy people. This fails the first postulate (depending on how loosely you apply 'should'). 

What about the same agent in different species? The dogma with Bluetongue virus is that it is asymptomatic in cattle, but can be lethal in sheep. Even with sheep though, only a few of those which become infected actually show bluetongue disease. The fact that the term 'case fatality' exists is an acknowledgement itself that only a fraction of animals which become infected fall ill. You could argue that this is an irrelevance as, even if it doesn't cause disease in every animal that it infects, it is still the causative agent of those which do become sick. Even though there haven't been enormous numbers of cases reported (presumably because there's no evidence of disease), I would put a lot of money on the fact that a large proportion of cows in southern England are currently seropositive for Schmallenberg virus.

In turn, when attempts are made to fulfill postulate 3, it would be perfectly normal, expected even, that an animal doesn't become sick.   

Discussions of these limitations are not new. The biggest development that has prompted the questioning of Koch's postulates has been molecular biology. The extreme sensitivity of nucleic acid-based methods means that evidence of pathogens can be detected at extremely low levels. Now it is possible to go searching for viruses with relative ease, increasing the speed and efficiency of virus detection and discovery. There are many approaches; PCR is arguably the most common, and metagenomics the most recent. I compare the methods to fishing: firstly, it’s possible to go fishing for specific viruses using PCR. Metagenomics on the other hand is more like a deep sea trawler – sequencing everything within a sample and looking within the ‘catch’ for virus sequences. Using the latter approach has allowed the identification of numerous viruses, and this is reflected in the number of publications, as revealed in a paper by Mokili et al. (2012).

Viral metagenomic studies between 2002 and 2011. From Molili et al 2012.

The paper by Mokili et al. (2012) also discusses the fact that molecular approaches remove the need for the growth of an agent in pure culture. Significantly, there is also the acknowledgement that determining causality merely by a pathogen’s presence is difficult to achieve, particularly with metagenomics approaches which are capable of finding a diverse array of agents within the same sample. The authors go on to describe a ‘metagenomic Koch’s postulates' approach whereby the metagenomes of individuals are compared. The postulates are:

1) The diseased metagenome must be significantly different from the healthy control and contain a greater abundance of the suspected metagenomic traits.
2) Inoculation of a healthy individual with a sample from the diseased individual must result in disease state.
3) Selected, specific samples containing the suspected traits from the individual infected for step 2 must cause disease when injected into another healthy individual.

Metagenomic Koch's postulates as described by Mokili et al 2012. Comparison between a diseased and healthy control animal shows a significant difference between the metagenomic libraries (depicted by the histograms of relative abundance reads). In order to fulfill the metagenomic Koch's postulates: (1) The metagenomic traits in diseased subject must be significantly different from healthy subject. For example traits A, D, E and J found in the disease animal that are not present in the healthy control; (2) Inoculation of samples from the disease animal into the healthy control must lead to the induction of the disease state. Comparison of the metagenomes before and after inoculation should suggest the acquisition or increase of new metagenomic traits (A, E and P). New traits can be purified by methods such as serial dilution or time-point sampling of specimens from a disease animal. (3) Inoculation of the suspected purified traits into a healthy animal will induce disease if the traits form the etiology of the disease

Following this procedure allows the sequence associated with disease, i.e. a biomarker of the etiological agent, to be discovered by the process of elimination.

Metagenomics will, rightly, more than likely become established as the method of choice for diagnosis. The technology is still developing, but not too far in the future I suspect metagenomics will follow a similar path to that of real-time PCR into the molecular diagnostics setting. Instead of testing a samples against some 'likely suspects' using PCR approaches, it will be possible to get a complete picture of the complex 'virome' associated with that sample.

Although the 'metagenomics Koch's postulates' are a step towards linking metagenomics to disease, there is still the issue of ‘must cause disease’; if this ‘disease’ by definition involves clinical signs, then similarly to Koch’s original postulates this may fail. However, the approach does allow the picking apart of causality in complex scenarios where multiple pathogens are present. There are still issues to be ironed out as to how molecular data is interpreted, but this is going to become increasingly important as metagenomic approaches become even more widespread. From this perspective, it appears that Koch’s postulates, landmarks and revolutionary as they may have been, may be nearing the end of their life.

Mokili, J., Rohwer, F., & Dutilh, B. (2012). Metagenomics and future perspectives in virus discovery Current Opinion in Virology, 2 (1), 63-77 DOI: 10.1016/j.coviro.2011.12.004

Sunday, 25 November 2012

When is a zoonosis not a zoonosis?
Zoonosis. One of the current glam terms in virology. At it's simplest, a zoonosis refers to a disease that is transmitted to humans from animals.

But it's possible to pause and think, what actually counts as a zoonosis?

On the WHO website it's classed as "any disease or infection that is naturally transmissible from vertebrate animals to humans and vice-versa".

From Wikipedia "an infectious disease that is transmitted between species (sometimes by a vector) from animals to humans or from humans to animals".

From the medical dictionary....."refers to diseases that can be passed from animals, whether wild or domesticated, to humans".

In all of these definitions the key word is 'disease'. Look it up and disease suggests there's some sort of pathology, with overt symptoms - admittedly this too is wide open for discussion. Taking this forward, if an animal virus infects a human, but doesn't cause disease, can that then be classed as a zoonosis?

Dealing with an outbreak of Hendra virus; passed from bats to horses...and on to the handlers

I was told once that, many years ago, people who mouth pipetted viruses were seropositive for the (livestock) viruses they were working on. If there were symptoms, they weren't sufficiently serious to become part of the story, so in this case we might argue that there was no disease. That fits with the definition - no disease, therefore not a zoonosis - and indeed these viruses are not regarded as being zoonotic.  

Mouth pipetting; no longer a method of choice

An interesting paper which has recently been accepted into the Journal of Virology describing the isolation and characterisation of two novel paramyxoviruses, Achimota virus 1 and Achimota virus 2 (genus Rubulavirus) from bats (Baker et al., 2012). I enjoy a bit of virus discovery and it's nice to see a study done well with a good level of characterisation and epidemiology. The authors collected urine samples from under an Eidolon helvum bat roost and added them to cultured cells in the lab. Using the viruses recovered from the isolations they could then work out the seroprevalence, the results of which showed that there was evidence of the virus infection in E. helvum samples throughout the geographical range of this species. Variations in seropositivity among different age groups, and from year to year also showed that there has been active circulation of the virus. 

Bizarrely, one of the most intriguing things was the title, namely the "potentially-zoonotic" bit. The genus Rubulavirus contains some serious viruses, notably mumps virus, so this is an important point. The authors speculate about the zoonotic possibility based upon serology, where they found 3 people out of 442 to have evidence of prior exposure to the virus. 

But does this constitute a zoonosis? One of the positive samples was from a febrile patient, so perhapsThe authors acknowledge that whether or not these viruses are zoonotic will take some nailing down. As discussed above though, seroconversion against a livestock virus does not lead to the livestock virus becoming classed as zoonotic. So does the finding of 3/422 being seropositive for these viruses mean that they are zoonotic? Is seroconversion really sufficient to class something as zoonotic? 

Baker, K., Todd, S., Marsh, G., Crameri, G., Barr, J., Kamins, A., Peel, A., Yu, M., Hayman, D., Nadjm, B., Mtove, G., Amos, B., Reyburn, H., Nyarko, A., Suu-Ire, R., Murcia, P., Cunningham, A., Wood, J., & Wang, L. (2012). Novel potentially-zoonotic paramyxoviruses from the African straw-colored fruit bat, Eidolon helvum Journal of Virology DOI: 10.1128/JVI.01202-12

Tuesday, 13 November 2012

Crystal meth....a new way to treat influenza?
It wouldn't be unreasonable to suspect that smoking methamphetamine, one of the most widespread and damaging illicit drugs, would lead to enhanced susceptibility to various respiratory pathogens, such as influenza. That's what Chen et al had in mind when they set out to see whether meth had such an effect on influenza replication in cell culture; after all, it's already associated with enhanced susceptibility to other pathogens such as HIV and HCV (due to biological, as well as behavioural, factors). It seems this may not be the case. The study used levels of meth which are likely to be found in meth users blood, so in that sense it's realistic. When they tried infecting cells pre-treated with meth with influenza A virus, whilst the virus was able to replicate, it didn't reach the levels of control cells which hadn't been treated with meth. Similarly, when they looked for the expression of viral proteins in infected cells, they found that, as the concentration of meth increased, the level of viral protein decreased, further showing that meth is detrimental to influenza replication in this system.

Treating cells with increasing amounts of meth resulted in a does dependent reduction in the expression of the viral proteins M1 and NS1.

So how does meth affect the influenza virus lifecycle? Treating the virus with meth and then infecting untreated cells didn't make a difference to the number or size of the plaques which were formed, suggesting that meth doesn't affect the ability of the virus particles to infect and replicate in the cells. Therefore it's presumably downstream of entry; extrapolating to a human, meth therefore might not prevent the chances of becoming infected. Indeed, the study found the inhibitory effects to occur during the actual replication. Interestingly, the inhibitory effect doesn't appear to be significantly linked to enhancing the interferon response.

Treating influenza virus with meth didn't alter the development or size of plaques
This all leads to the tongue in cheek suggestion that if you catch influenza then smoking a bit of crystal meth may help treat the infection. Perhaps it does, and it will be intriguing to see whether the method of action can be determined and therefore less damaging drugs discovered, but that would not justify consuming meth, one of the most ravaging and repulsive drugs around. For a start, one of the stark realities about this paper is that it is using cultured cells, which doesn't really mimic the complexity of the respiratory tract. Then there are the effects of the drug: enhanced susceptibility to other pathogens, addiction, depression, heart disease, anxiety, 'meth mouth', altered heart and breathing rates, diahorrea, constipation, insomnia, hallucinations etc. etc.........

Meth mouth

......anyone fancy a cohort study???

         Chen, Y., Wu, K., & Chen, C. (2012). Methamphetamine Reduces Human Influenza A Virus Replication PLoS ONE, 7 (11) DOI: 10.1371/journal.pone.0048335

Saturday, 6 October 2012

Crimean-Congo Haemorrhagic fever virus comes to Glasgow

This week saw Glasgow's first case of Crimean-Congo Heamorrhagic Fever virus (CCHFV), a virus belonging to the family Bunyaviridae which can cause (as the name suggests) a haemorrhagic disease. 

A man arrived from Dubai, having been bitten by a tick in Afghanistan, and a couple of days later went to hospital where it was confirmed that he had CCHFV. Sadly news has just surfaced that he's died, highlighting what a severe virus this can be. Headache, muscle and joint pains, vomiting, diarrhea, bruising and bleeding are all associated, to a lesser or greater extent, with CCHFV, along with a fatality rate of up to 30%.

CCHFV is a tick-borne virus which is hugely widespread; the rather bizarre name reflects its discovery in both Crimea and Congo at (roughly) the same time. Eastern Europe, large swathes of Asia and the Indian subcontinent and Africa are all affected by, or at least have some evidence of, CCHFV. 

The world distribution of Crimean-Congo Haemorrhagic fever virus
Perhaps it's their relative unfamiliarity compared with mosquitoes and midges, perhaps it's the way they lock on to a host for extended amounts of time, but there always seems something rather repulsive about ticks. They seem to be everywhere, but not all seem to be infected by CCHFV, which tends to infect species of the genus Hyalomma. If it's not via a tick bite, then an alternative way to become infected is contact with the blood of an infected animal. But if it's not endemic to western Europe and the UK, would it ever be a problem here? Can it infect the ticks here? A few years ago people thought it would be more or less unthinkable that Bluetongue virus would ever reach northern Europe and the UK, an assumption dismissed emphatically in the last few years. Similarly, Schmallenberg virus is another arbovirus that has swept across Europe (incidentally, SBV is from the same family of viruses as CCHFV).

Is it possible that it would ever establish itself here? I'd guess it's pretty unlikely right now. But with climate change, species of tick that are known to be capable of being infected by CCHFV might be on the move, and based upon the climates in other areas, the climate of western Europe might just suffice.  

Friday, 28 September 2012

Badger Herpes
For anyone reading this outside of the UK a huge uproar is currently occurring over the imminent start of a trial whereby it will be legal to intensively cull badgers. The hope is that this will reduce bovine TB (bTB) which is rife in 'hotspots' in the south west of England and Wales. The controversy is that a previous culling trial, the Randomised Badger Culling Trial (RBCT), suggested that reactive culling (that is, culling badgers in an area where a bTB breakdown has occurred) disrupted badger communities with the result that bTB incidence increased (incidentally, this itself adds further weight to the fact that badgers are important in bTB epidemiology). However, in areas where culling was proactive (whereby all badgers in an area were culled up front), bTB levels did indeed drop by a significant level (Vial and Donnelly, 2012), so culling works, right? The current trial is aiming to create regions which have been culled to a sufficient extent to reduce bTB incidence by culling within boundaries to badger movement (rivers, busy motorways etc.). Interestingly, the results of culling in Ireland are somewhat more positive, and in previous periods of culling in England (intensively using gassing) bTB levels were low. 

The large proportion of the UK population like badgers and, combined with the contrasting results, there are plenty of people who are less than keen for the culling to go ahead (hopefully they won't cause the level of culling to be insufficient and thereby cause an increase in bTB!).

Brian May is a big fan of badgers.
So what about viruses? A paper by Banks et al (2002) described the discovery of a gammaherpesvirus in a badger from Cornwall in England. The paper reports bits of sequence from the virus; nowadays the complete genome would, in theory, be relatively simple to obtain and report. The badger was negative for bTB (based on histological and immunological examinations) but was in a poor state of health and had pathology in the liver, kidneys and lungs. Of course this doesn't mean it was the virus that caused these effects. It would be interesting, and potentially important, if this virus inadvertently interferes with bTB epidemiology.
According to the phylogeny, badger herpesvirus appears to fit nicely among other herpesviruses, most closely with equine herpesvirus 2.

Bager herpresvirus (BadHV) most closely related to equine herpesvirus in either the gb  (a) or  DpoI (b) genes. From  Banks et al 2002

The virus can be propagated in mink cells so in theory could be manipulated, though my current knowledge of herpesviruses isn't that great. Perhaps a virological-fantasy would be that the virus could be modified in such a way that it induces immunity/resistance to bTB in badgers - release the virus into the wild badgers and let it spread through the population. Simple and effective. 

But releasing a genetically modified virus into the wild......would that really be any less controversial than the culling?

Banks M, King DP, Daniells C, Stagg DA, & Gavier-Widen D (2002). Partial characterization of a novel gammaherpesvirus isolated from a European badger (Meles meles). The Journal of general virology, 83 (Pt 6), 1325-30 PMID: 12029147
Vial F, & Donnelly CA (2012). Localized reactive badger culling increases risk of bovine tuberculosis in nearby cattle herds. Biology letters, 8 (1), 50-3 PMID: 21752812

Sunday, 16 September 2012

Virus evolution: hitting the bottle(neck) in mosquitoes
When a virus population transmits between hosts, regardless of how this is achieved, not all of the progeny viruses make it, most likely the majority are left behind to contemplate a life of non-existence and the impeding destruction. At the animal level this is fairly straightforward; an animal becomes infected, produces virus, some of which will infect a new animal, which then becomes infected etc. etc. But transmission events impose bottlenecks. Imagine a mosquito taking a bloodmeal from a human. The virus population within that person will, more or less, be spread throughout the body. An adult human contains approximately 5 litres of blood; a mosquito will only take a few microlitres, i.e. a few millionths, of the blood, and therefore only a tiny fraction of the virus in the body. Taken to a more subtle level, of the virus which makes it to a new host, only a fraction of that virus will infect cells within the new host. In the case of the mosquito, there are many 'barriers' which are familiar to virologists studying arboviruses. The first step towards infecting a mosquito (or other biting insect such as midges) is to infect the midgut. At this point there's another bottleneck, known as the midgut-infection barrier; only a few of the viruses which were taken up in the blood-meal will be able to infect the gut cells. In the case of bluetongue virus in midges there are other described barriers, including a midgut escape barrier, a disemmination barrier, and a salivary gland infection barrier. All of these must be overcome in order to allow transmission to another mammalian host.

An array of barriers must be overcome by a virus in order to continue the virus lifecycle. Image from Black et al, 2002. 

The significance of this becomes more apparent when the virus population itself is considered. Viral RNA polymerases are notoriously error prone, resulting in a population of viruses with differences in their genomes, in other words the so-called quasispecies (itself something worthy of discussion). What happens to the population structure as the transmission cycle progresses through the various stages and bottlenecks?

An interesting talk at the SGM in Dublin this year has now been published in PLoS Pathogens. In this case, the authors made an artificial virus population of Venezuelan equine encephalitis virus (VEEV) using reverse genetics. This then allowed particular clones to be followed through the various steps of infection as the virus passes from a mouse containing the clones through to how many are injected into a fresh mouse at the end of the mosquito steps of the virus lifecycle.
If they allowed mosquitoes to feed on a mouse containing the different 'clones' of VEEV, and then looked to see how many they could find in the mosquito, they found between 6 and 8 of the clones (i.e. ~70% of all clones). After a few days however, once the infection process is under way, the number of clones had decreased, implying that not all of the clones had made it through the midgut infection process.

By the time the virus has disseminated to the legs/wings, and salivary glands, there were only 1-4 of the clones present, suggesting that yet more had been lost along the way. In the saliva, which represents the population of virus that will be passed on to the new host, they only found on average 1-3 of the clones. This contrasts to the number of clones found in the legs/wings, showing that only some of the clones from the haemocoel managed to successfully infect the salivary glands.

Interestingly, there didn't appear to be a strong bottleneck in transmission to an uninfected mouse; the same clones that were in the saliva were the same clones which infected the mice, although considering the low numbers of clones in the saliva there wasn't a massive choice.

All of this leads to the puzzling question of how does the virus survive? If at each point viruses are removed, then by chance the surviving population may be less fit. Looking for marked viruses as this study has done is an elegant way of doing it, but it is limited to signatures within the genome. A further study using deep sequencing to look at the whole population more comprehensively may offer even more detail about what's going on. Perhaps this all relates to the observation of purifying selection being popular among arboviruses, time will tell, but to begin with this study has shown that such barriers exist. How the severity of the barriers vary, and how the virus deals with these barriers, will be interesting to see.

William C. Black IVa, , , Kristine E. Bennetta, Norma Gorrochótegui-Escalantea, Carolina V. Barillas-Murya, Ildefonso Fernández-Salasb, Marı́a de Lourdes Muñozc, José A. Farfán-Aléd, Ken E. Olsona, Barry J. Beaty (2002). Flavivirus Susceptibility in Aedes aegypti Archives of Medical Research DOI: 10.1016/S0188-4409(02)00373-9

Thursday, 30 August 2012

African Swine Fever on its way, (though not from Africa)

Sometimes I think the numbers quoted in the introductions to papers and presentations etc. are a little inflated. Certain strains of Bluetongue virus, such as BTV-8 (Europe) or BTV-17 (US) can result in a high case fatality of around 40%, and this is what's often quoted, but more usually the fatality rates are much much lower. H5N1 'bird 'flu' is certainly dangerous, but Vincent Racaniello recently highlighted that this might not necessarily be the whole story. There are though exceptions where the fatality rate is said to be high, and is high; African horse sickness, for example, never seems to be much less than 50% fatal, which is not great if you're a horse owner. African swine fever is another virus which fits into this latter 'always nasty' category. 

I've heard about the devastation caused by ASFV from a Ghanaian vet who said he had been to farms in Africa where every pig was dead. If you're a pig farmer you've got to be worried; sheep farmers losing lambs to Schmallenberg virus have had a hard time, but if 100% of your stock die, what then? Imagine a barn full of dead pigs, it can't be great.
Dead pigs as a result of African Swine Fever Virus

ASFV is a big DNA virus that's transmitted by soft ticks. In Africa the virus persists in the environment with its wild host, wart hogs. In Europe though, it has the option of persisting in wild boar, of which there are many.

ASFV has historically been linked, not surprisingly given the name, with sub-Saharan Africa. Before 1957 that's where it stayed. Since then there have been occasional introductions into Europe, and more or less has always been eradicated. That is, until 2007 when there was an outbreak of ASFV in Georgia. Since then the virus seems to have been hanging around in the region, with the odd report here and there of a new outbreak in the Caucasus. Now though, for the first time ever, it's in Ukraine. It's on the move and, rightly, people are worried. Thus far, the Middle East, where there aren't many pigs, has provided a buffer to ASFV encroachment into Europe through Turkey. Now though there is the possibility of the virus coming in via a different route into areas where the pig density is much greater.

In a forest near you: will AFSV-infected wild boar soon be roaming Europe?

Infected meat has always been a threat for ASFV introduction to the UK, although regulations and the restrictions on swill feeding help curtail this threat. However, wild boar don't respect international boundaries, so if the virus manages to get into the tick population, and then wild boar, it could result in a tricky situation where it's difficult to weed out the virus. Wild boar were recently at blame for an outbreak of Foot and Mouth Disease virus in Bulgaria, hopefully they won't be responsible for introducing ASFV.   

Thursday, 16 August 2012

So what, the virus fluoresces; who cares?
The day after I infected the cells I went to the fluorescence microscope and looked for fluorescence. The uninfected control? Nothing, black. The culture with cells infected with BTV-mCherry had islands of red. It had worked. I had made a bluetongue virus which, when it infects cells, expresses a fluorescent protein, in this case mCherry (one of many options - a girl working with me wanted mPlum).

BTV-mCherry replicating in a Drosophila fruit fly. Green fluorescence represents autofluorescence of fly tissues.
Red =  fluorescence generated upon BTV-mCherry infection.

But why bother? What use is a virus which makes cells light up?

The reason it came in useful for us is that we wanted to see where in an insect the virus replicated. A more straightforward approach would be to find the virus using antibodies and then reveal the antibodies - immunolabelling. But this resulted in the insect muscles fluorescing. Using the fluorescent virus meant that we didn't need immunolabelling; instead we simply sliced up the insect and looked at which bits lit up when illuminated at the wavelength for mCherry.

Another reason is so that virus infections can be filmed - live cell imaging - to reveal dynamic processes. For example, virus protein 'X' is tagged with a green molecule. Where does it go? Fixing the cells means looking at just a point in time, rather than 'the virus protein travels from here (A) to here (B)'. A fluorescently tagged virus therefore lets you film the virus in real-time. I saw this in action with a fluorescently labelled Rinderpest virus (prior to its eradication). As the virus spread it formed large syncytia, swallowing up cells in an ever growing expanse of green. 

Cells infected with GFP-expressing Rinderpest virus from Banyard et al., (2010). A; individual cells.  B; syncitia.

The ability to film dynamic processes of an infection has other advantages. Fluorescence can be measured, and can be used to reflect the extent or rate of replication: in addition to its use in virus research, this can obviouly be used as a measure of therapeutic value.

And in the animal?
The simplicity of looking for these sorts of viruses in animal tissue means it's possible to sample from more tissues of the infected animal, increasing our knowledge of the virus in the host. Even during infection, a machine can sort a blood sample so that we can find which blood cells the virus is replicating in. The insect infected with bluetongue is one example, but the most impressive I've seen so far was at the SGM in Dublin (now published in J. Virology). A canine distemper virus expressing GFP (green) or dTomato (red) was used to infect ferrets. This time it wasn't just a case of looking at dissected tissues, the authors looked at entire organs to determine where the virus replicated: virus infection at the macroscopic level - something impossible without such a virus.

Ferret tissues infected with green or red expressing Canine distemper virus (from Ludlow et al, 2012); A, infection around the eye and mouth, but not the nose; B, gingiva; C, skin epidermis; D, tongue and tonsils; E, salivary gland and lymph nodes; F, lungs; G, liver; H, spleen; I, stomach/gastrointestinal tract; J, infected B cell follicles in Peyer's patches; K, absence of fluorescence in the leptomeninges (redCDV); L,  fluorescence in the leptomeninges (greenCDV).

So, there you have it, not so useless after all. As freaky as they may be, fluorescently tagged viruses certainly have their place.

Shaw AE, Veronesi E, Maurin G, Ftaich N, Guiguen F, Rixon F, Ratinier M, Mertens P, Carpenter S, Palmarini M, Terzian C, & Arnaud F (2012). Drosophila melanogaster as a Model Organism for Bluetongue Virus Replication and Tropism. Journal of virology, 86 (17), 9015-24 PMID: 22674991

Banyard AC, Simpson J, Monaghan P, & Barrett T (2010). Rinderpest virus expressing enhanced green fluorescent protein as a separate transcription unit retains pathogenicity for cattle. The Journal of general virology, 91 (Pt 12), 2918-27 PMID: 20719989

M. Ludlow, D. T. Nguyen, D. Silin, O. Lyubomska, R. D. de Vries, V. von Messling, S. McQuaid, R. L. De Swart and W. P. Duprex (2012). Recombinant Canine Distemper Virus Strain Snyder Hill Expressing Green or Red Fluorescent Proteins Causes Meningoencephalitis in the Ferret Journal of virology, 88 (14) DOI: 10.1128/JVI.06725-11

Wednesday, 8 August 2012

Schmallenberg....where are we at?

There's increasing chat about the circulation of Schmallenberg virus in the UK this summer. Is that surprising? Maybe, it was more or less impossible to know as the virus was new. Likewise, new virus, new test. Serology (looking for antibodies as evidence that an animal has previously been infected) is the way to find where it's been and there's a commercial ELISA to do this serology. But how can you compare it to a gold standard when there isn't a gold standard? There's always the chance that the test doesn't detect as many as it should; the bTB skin test is the classic case of a test being hopeless. 

An ELISA showing cattle sera positive for SBV antibodies

Is the circulation important? The worrying thing is that we only have a rough idea about prevalence. The situation is reminiscent of the situation in Germany in 2007; seeded with bluetongue virus in late 2006, BTV exploded in Germany the following summer, with 1000s farms being infected. The main indication of SBV infection seems to be congenital deformities, so, unlike BTV, we'll have to wait; it could still be the horror everyone was worried about.

Re-circulation does bring it onto the radar of vaccine companies though, so vaccines might be available soon, but when exactly nobody knows. One of the ideas with Akabane virus (one of SBV's close relatives) is that protection is achieved young and thus the animals may be more resistant to infection when pregnant. 
So is Schmallenberg important? We still don't know. If it affects fertility it could be massive. Right now  though it's 'watch this space'. Certainly in the south west of England, bTB remains the major scourge.

Tuesday, 17 July 2012

The Wolbachia story rolls on
For the last few years one of the hot topics in arbovirology has been Wolbachia, an endosymbiotic bacterium of insects. One aspect of Wolbachia is that it can affect insect reproduction, but in the case of viruses the interesting fact is that infection with Wolbachia can lead to resistance to infection by viruses.

Initially the observations were made in the fruit fly Drosophila melanogaster and the induction of resistance to a variety of RNA viruses upon infection with Wolbachia. This was quickly seized upon as a potential tool for altering the transmission potential of mosquito populations; infection with Wolbachia would effectively reduce the vector competence of mosquitoes to transmit pathogens. In turn, the last couple of years have seen the successful infection of notorious mosquito species such as Aedes aegypti and A. albopictus with Wolbachia. As was predicted, the Wolbachia infection led to the induction of resistance to infection of the mosquito by some medically important viruses such as dengue virus (Bian et al., 2010) and Chikungunya virus (Mousson et al., 2010). We recently showed that fruit flies infected with Wolbachia are resistant to infection by Bluetongue virus (Shaw et al., 2012).

This work has got to the stage where field release of the mosquitoes are a real possibility, it will be interesting to see whether there are similar successes to some of the well known sterile male releases. 

It was interesting therefore that a paper came out recently describing the infection of army worms (Spodoptera exempta) in Tanzania in which infection with Wolbachia resulted in increased susceptibility to infection with a baculovirus, S. exempta nucleopolyhedrovirus (Graham et al., 2012). At present there's not really much idea why this is. Clearly it's a different system; the insects are Lepidopteran rather than Dipteran, and the virus is a dsDNA virus as opposed to the RNA viruses which have been studied so far. Different dynamics are clearly at work and it will be interesting to dissect the interactions between the insect, Wolbachia and the virus to determine the reason behind the differing outcome. 

There is, however, another important message. The fact that this system differs from that observed with mosquitoes/Drosophila and RNA viruses should be a clear signal that things are often more complicated than it may appear, and it would be dangerous to assume otherwise. It's reminiscent of the situation with   Bluetongue virus. Until the BTV-8 outbreak in Europe a few years ago, where it was found that the virus could pass the placenta and infect the fetus, the ability of BTV to cross the placenta was thought to be strictly a property of cell culture adapted live vaccine strains. BTV-8 proved this not to be the case. Similarly, the Culicoides midges which transmit BTV were thought not to go inside buildings, so one of the recommendations for protecting your animals was to keep them indoors when midges were active outdoors. When the outbreak happened in northern Europe, people started looking at the local midge species and found that, actually, they do go into buildings. 

Clearly there's not always such a thing as 'one size fits all'.

Bian G, Xu Y, Lu P, Xie Y, & Xi Z (2010). The endosymbiotic bacterium Wolbachia induces resistance to dengue virus in Aedes aegypti. PLoS pathogens, 6 (4) PMID: 20368968

Mousson L, Martin E, Zouache K, Madec Y, Mavingui P, & Failloux AB (2010). Wolbachia modulates Chikungunya replication in Aedes albopictus. Molecular ecology, 19 (9), 1953-64 PMID: 20345686

Shaw AE, Veronesi E, Maurin G, Ftaich N, Guiguen F, Rixon F, Ratinier M, Mertens P, Carpenter S, Palmarini M, Terzian C, & Arnaud F (2012). Drosophila melanogaster as a model organism for bluetongue virus replication and tropism. Journal of virology PMID: 22674991

Graham,R.I., Grzywacz,D., Mushobozi,W.L. and Wilson,K. (2012). Wolbachia in a major African crop pest increases susceptibility to viral disease rather than protects Ecology Letters DOI: 10.1111/j.1461-0248.2012.01820.x

Monday, 25 June 2012

Bee vs. Virus. Who decides? One of the most frequently occurring quotes going around referring to bees is "If the bee disappeared off the surface of the globe then man would only have four years of life left. No more bees, no more pollination, no more plants, no more animals, no more man," (often attributed to Albert Einstein). Regardless of whether or not this came from Einstein, the idea is still one worth thinking about. Honeybees (Apis mellifera) are often the most significant pollinators of plants, so their disappearance would be something of a concern.

Bees face many challenges. More and more people have heard of colony collapse disorder (CCD), a phenomenon whereby colonies of bees seem to suddenly die out. The most infuriating aspect of CCD is that the absolute cause is unknown; whether it’s biotic factors such as viruses, chemical factors, such as pesticides or even simply a lack of nutrition. There are many diseases of bees, including numerous viruses, but the most significant issue bees (and their beekeepers) face is Varroa destructor; a small mite which has caused immense problems wherever it’s ventured.   

A couple of papers have come out recently regarding the demise of bee colonies, both concentrating on the interactions between infestation of a colony with the Varroa mite and Deformed Wing Virus (DWV), a small RNA virus of the family Iflaviridae.

The first study was published in Science and benefits from studying the ‘before and after Varroa’ scenarios, as opposed to studies of colonies in regions already infested with the mite. The scientists compared Hawaiian islands with and without the mite, and then again once the mite had spread to other islands. Firstly they found that over the years Varroa prevalence increased. In turn, as Varroa levels increased, the infection levels of DWV in each bee also increased, whilst the diversity of the DWV strains infecting the colonies reduced. Effectively the Varroa mite had rendered a relatively resistant population susceptible to DWV and as a result DWV exploded through the population.

The Deformed Wing Virus relationship to Varroa; infestation with Varroa allows  DWV to explode.

A second study, published in PLoS Pathogens, found a similar result; as the extent of Varroa infection increased, so did the levels of DWV. Whereas the Hawaiian study observed the situation on a grand scale, the authors of the PLoS looked further into the interactions and found evidence that allowing mites to feed on bee larvae resulted in an increase in DWV levels. When they looked into the effect of the virus on the bee’s immune system, they observed that the expression of certain genes relating to bee immunity altered in response to infection by DWV. Essentially, bees infected with DWV were immunosuppressed, but as a result of DWV, not Varroa feeding on the larvae. Instead, it would appear that, by trying to repair the feeding holes made by Varroa, the bee inadvertently weakens its resistance to DWV, thus promoting DWV replication.

These studies add to the growing amounts of data linking Varroa infestation and DWV as a cause of colony collapse. They also highlight how it’s possible to view a colony as an organism itself, rather than individuals; things such as Varroa affect the colony as a whole, and, ultimately, it is the colony that collapses. 

< Nazzi F, Brown SP, Annoscia D, Del Piccolo F, Di Prisco G, Varricchio P, Della Vedova G, Cattonaro F, Caprio E, & Pennacchio F (2012). Synergistic parasite-pathogen interactions mediated by host immunity can drive the collapse of honeybee colonies. PLoS pathogens, 8 (6) PMID: 22719246

Thursday, 31 May 2012

Bat rabies virus evolves at rate X....or does it?

One of the most interesting aspects of virology is the obligatory dependence of the virus upon its host. This dependence means that viruses have an intricate relationship with their host and have evolved to utilise the components of a specific host, although many viruses are happy to replicate in other organisms. For some it’s a necessity; arboviruses such as West Nile virus (WNV) must replicate in both vertebrate and invertebrate (in the case of WNV a mosquito) hosts to complete their lifecycles. Inevitably, this means that viruses can jump the species barrier leading to new (and potentially dangerous) scenarios where viruses behave differently in the new host. HIV for instance is thought to originally have crossed the species barrier into humans from chimpanzees.
Different hosts means different evolutionary pressures on a virus; factors such as bottle-necks and rates of transmission as well as immunological pressure will all impact upon the virus. A recent paper in PLoS Pathogens looks at how rabies virus evolves at different rates in different species of bat, and how the biology of bats impacts the subsequent evolution ofthe virus.

It seems that, once a virus is established in a particular species, the evolutionary rate remains fairly constant, although some virus lineages were able to evolve at a rate 5-22 times greater than that of others. Whilst the virus lineages themselves can show variation, they looked at whether there was variation in the rates of evolution in different species of bats and their ecology. Interestingly, the slower evolving lineages tended to be those evolving in bats of temperate zones, where the viruses were found to evolve at a rate nearly four-fold lower than that in tropical and sub-tropical areas. It seemed that the rate of evolution correlated not with a particular bat, but with where the bat was living. So do colder temperatures mean a lower metabolic rate and thus a reduced virus evolutionary rate? Apparently not. It appears it’s probably more to do with transmission rates and the impact that this has upon virus evolution.

Rabies virus evolves slower in species of bat dwelling in temperate areas

This all reminds me of a paper last year showing that different West Nile virus genomes predominated in different hosts (vertebrate vs. invertebrate) and that this affected the fitness of the virus in the alternative host (1). All in all, these kinds of study show just how complex viral evolution can be; can this all be explained by an evolutionary model based upon the concept of quasispecies?

(1) J. Virol. 2011. 85(23)12605-13

Streicker DG, Lemey P, Velasco-Villa A, & Rupprecht CE (2012). Rates of viral evolution are linked to host geography in bat rabies. PLoS pathogens, 8 (5) PMID: 22615575

Thursday, 24 May 2012

Learning from (virus induced) mistakes

One of my favourite popular science books is ‘Mutants: On Genetic Variety and the Human Body’. In the book Armand Leroi describes various humans which, although at the time may have ended up in one of those infamous ‘freak shows’, have a selection of different deformities of varying severity. The book is illuminating in that it follows the idea that if we can see what made something go wrong during development, then it tells us something about what happens when things go to plan. Knowing what we do now about development, it is possible to look back at these historic cases and explain them biologically. For instance, if someone is born with a deformed limb, and we find that they have a mutation in a specific gene, we can say that the gene is somehow involved in limb development.

Mutations are natural. As Leroi sates, “we are all mutants, but some of us are more mutant than others”. However, they can also be induced. Studies of embryogenesis and development are inextricably linked with Drosophila fruit flies. Many facets of development were figured out by inducing mutations in Drosophila with X-rays, resulting in a bewildering number of mutants being described with diverse and imaginative names. Individual pathways and genes have since been correlated with particular phenotypes.

Antennapedia mutant of Drosophila: flies possess legs in place of antennae

Not all errors in development are directly linked to mutations. 1957-1961 saw the tragic incident of thalidomide, a drug taken by pregnant women to ease the discomfort of morning sickness. It soon became apparent that thalidomide was teratogenic with the resulting offspring sometimes possessing, among other deformities, severely stunted limbs. Clearly the presence of thalidomide interferes with foetal development. Although it is still not fully understood, various ideas exist about the way in which it causes the effects, including angiogenesis in the developing limb, thus stunting outgrowth (1).

A mother sheep sniffs her Schmallenberg virus-infected offspring (Kreisverwaltung)
A ewe investigates her Schmallenberg-affected lamb
When all of the images of lambs with arthrogryposis started to emerge a few months ago as a result of Schmallenberg virus infection during gestation, it reminded me of the approach ‘Mutants’ was following. As a Bunyavirus, Schmallenberg virus only has 6 proteins. By looking into the pathways which these proteins disrupt it could be possible to learn something about proper limb development. Whilst looking into this a bit more, a paper has been published detailing the infection of foetuses with Cache Valley virus (2), another Bunyavirus which also causes congenital abnormalities in sheep.

The paper looks at the localisation of virus infection and the time-frame of infection and subsequent development of the malformations. The data support previous work revealing that infection of the central and peripheral nervous systems is responsible for reduced foetal movement and the eventual development of the arthrogryposis. This confirms the idea of using viruses to investigate development: observation of the tropism of the virus and correlating this to the deformities of the resulting offspring allows, at a somewhat macroscopic level, identification of the role of particular tissues in development. As more and more methods are developed it may be possible to elucidate the molecular pathways which are disrupted by infection with these viruses, thus linking development to a specific pathway. Clearly the results are likely to be more ‘pointers’ of what’s happening as opposed to precisely defined interactions, but learning from mistakes is always a good thing!

(1) Proc Natl Acad Sci USA. 2009. 106(21):8573-8.
(2) J. Virol. 2012. 86(9):4793-800