Field of Science

How to grow an awkward virus like Schmallenberg vrius?

If anyone works on viruses that naturally replicate in two distinct hosts and NEED to do this, maybe you can help here.

While having a look around for work on Schmallenberg virus (SBV), I came across a paper (this paper) that compares two methods of doing experiments with this virus in animals. On one hand they have infectious serum from cattle. That is they have blood which they have taken from a single cattle that had been infected with SBV and that is packed full of infectious virus particles. These can be injected back into another cattle for infection experiments. This I will consider mammal-mammal virus.

On the other hand they have cell culture isolated virus. This virus was taken from a cattle blood sample and grown on insect cell lines then grown on mammalian cells again and then from here it can be taken into animals. I will consider this cell culture (insect-mammal) virus.

Now based on animal experiments they say that the mammal-mammal virus, grown and harvested solely from infected cattle is 'better' to use than the cell culture (insect-mammal) virus. They say it is better based on looking at the infection kinetics following inoculation of 4 animals each, in particular it seems because that it replicates to a higher rate in cattle than the cell culture one. Despite not being statistically significant I might add (!).

"this difference provides a clear indication of the pitfalls of culture-based production
of challenge inocula"

Now I really don't think this is correct to state. At least without the right evidence.

For one it is good to remember that these viruses have two natural hosts out there in the wild. At least TWO. Viruses are replicating in insects, then mammals, then insects, then mammals again... Although they can spread from insect to insect (I think) and from cow to calf (mammal to mammal), this last one is an evolutionary dead end. So 'real' SBV, whatever that is, must be that which has replicated in insects and in mammals. Growing virus in any one of these hosts for one time is bound to allow for adaptation to a single host that may prevent it from growing in the other. It is the classical evolutionary trade-off.

In experiments that will determine the development of vaccines and pathogenesis studies we want to work with what is 'real'. Not something that is essentially a lab artefact. Although the virus may replicate to a higher level in cattle, this might not be what's out their in the wild.

Now this brings me to my final point. We don't know which is best because we don't have the evidence. To determine which one is best we would have to compare it to the kinetics of a 'real' infection. Something which might be difficult to do given it's uncontrollable (in a scientific way) nature. One way to get away from this might be to sequence the genome of mammal-mammal viruses and compare them with insect-mammal viruses. They didn't do this.

Without these two pieces of evidence in hand I would want to stick with what Nature does. I would grow the virus in it's natural and medically relevant host species. Midges and cattle. This way we assure that we are working with something that is 'real'. Although it might be cheaper to sequence the damned things and see what's happening during growth on the different 'substrates'.

Notes on zoonotic rubulaviruses

I've been meaning to post about this paper for a while now and thought that nearing the end of the year would be a good time to clear my head of thoughts about it.








The paper I'm talking about is one that was published recently in the Journal of Virology (http://www.ncbi.nlm.nih.gov/pubmed/23152534), and concerns itself on the discovery, characterisation and epidemiological investigations of two bat viruses. It was carried out by a large team from the UK and around the world. The first author was Kate Baker.

These viruses, which they isolated themselves (yes! actually, physically have virus to study) were found in the urine of fruit bats (species: Eidolon hevlum, the straw coloured fruit bat - a truly magnificent animal). There's good evidence that these viruses are natural 'pathogens' of bats and there is limited evidence to suggest that there has been human exposure to them in the past. The importance of this is limited but personally I would prefer to know what is out there before it emerges.

For another take on this paper check out Andrew Shaw's Virus Musings blog: http://www.virusmusings.blogspot.co.uk/2012/11/when-is-zoonosis-not-zoonosis.html.

Antibiotic resistance found in isolated cave system

The next weeks #microtwjc paper has now been chosen and stuck up online over at http://microtwjc.wordpress.com/2012/12/09/microtwjc-week-17-christmas-edition-paper-and-discussion-points/ . If you want to discuss it, log in to twitter and join us Tuesday the 18th December at 8:00pm GMT. And if you are interested in the topic you should most definitely check this Nature review out http://handelsmanlab.sites.yale.edu/sites/default/files/AllenCalloftheWild.pdf

It's a neat paper that focusses on characterising the levels and kinds of antibiotic resistance in bacteria that live in a relatively isolated cave in New Mexico that has had extremely minimal human contact. The major point of this paper is that compared to other studies their site seems to be the most isolated microbial community, although this investigation in Alaska may be just as isolated. Although I dont think they can rule out water contamination from outside the cave system (actually from reading this article in NatGeo I think they can rule that possibility out). This they say is an 'ideal ecosystem' to study the original antibiotic resistance programs in the absence of human exposure.

To do this they employ a culture dependant approach, so obviously will only detect a small number of resistant microbes yet may be able to detect resistance mechanisms that we did not know about and so could not easily detect through purely molecular means. They do even find completely new ways that microbes have evolved to handle antibiotics.

One perhaps good thing about their results is that this cave is isolated so perhaps woudn't be such a reservoir for novel antibiotic resistance genes in a clinical setting.

A statement from their conclusion explains:

Antibiotic resistance is manifested through a number of different mechanisms including target alteration, control of drug influx and efflux, and through highly efficient enzyme-mediated inactivation. Resistance can emerge relatively quickly in the case of some mutations in target genes and there is evidence that antibiotics themselves can promote such mutations [43][44][45][46]; however, resistance to most antibiotics occurs through the aegis of extremely efficient enzymes, efflux proteins and other transport systems that often are highly specialized towards specific antibiotic molecules. Such elements are the result of evolution through natural selection; this therefore implies that antibiotic resistance has a long evolutionary past.

and

The remarkable genetic diversity of the antibiotic resistome, uncovered in this and other studies has additional practical application as an ‘early warning system’ for new drugs introduced into the clinic. Resistance mechanisms in the environmental resistome can emerge in the clinics and the clinical community should be aware of them...

Some questions I had are:

How isolated is this community?

Would it have better (possible?!) to sequence everything?

Should we be worried about this resistance?

If not effected by human antibiotic use, why do they have resistance mechanisms?

Antibiotic Resistance Is Prevalent in an Isolated Cave Microbiome

Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.

Meningitis B and the future of vaccines

This week is a good week for vaccines. Indeed it is a good week for society, at least in Europe, for we have just got word that the European Medicines Agency has approved Novartis's Meningitis B vaccine  and it could now be available in the UK as early as next year, if licensed here. *Novartis are currently working with the US authorities at getting it approved*


ResearchBlogging.orgThis vaccine, targeting Neisseria meningitidis group B bacteria (B subgroup causes the most problems in industrialised countries) is reported to be around 70% effective against the horrible and often deadly disease. It's roll out across the UK and Europe should save the lives and prevent the permanent damage that follows meningitis and septicemia. That's great, sure, but as scientists and people interested in public health, how the heck did they achieve this feat?

Mumps in New York - it's the size that matters

Two Jewish men in New York (Flickr by Kynan Tait). The site of 2009/10's near-4,000 large mumps outbreak

ResearchBlogging.orgIt started during the middle of 2009 when an eleven-year old boy returned home to the U.S from a holiday in the UK. The UK was just experiencing an exceptionally large (about 7,500 people) outbreak of mumps that year but don't worry, that boy had been twice vaccinated with the MMR immunisation. He should be OK. Shouldn't he?

As it turns out, he wasn't and he became infected with the mumps virus. And so our three year-long story begins.

The problem was that by the time this now mumps-infected boy realised he was ill (fever, swollen glands, potentially inflammation of the testicles and meningitis) he was attending a youth camp in New York along with 400 other orthodox Jewish boys. Unluckily for us, the mumps incubation period can be over 2 weeks and you can be infectious up to one week before this, making it particularly hard to contain as we will see.

A couple of days later the camp ended and each one of those mumps-exposed, potentially infected children were seeded back into society. By the time it had subsided nearly one year later, 3502 cases of mumps had been observed across the state of New York, the biggest outbreak the US had seen in decades and it was most likely attributable to this single index patient who brought the disease in from the UK.

But what was most worrying was that 76% of them had been immunised with two doses of the MMR vaccine, our mainstay of protection against the virus. So just how could mumps get passed this defence and cause such a massive outbreak? It took the U.S Centers For Disease Control and Prevention nearly 3 years to find an answer. The investigation is published in the New England Journal of Medicine here.

HIV in High-def

Plaque outside Antoni van Leeuwenhoek's old house in Delft
The dark age of microbiology existed in the years preceding Antoni van Leeuwenhoek's most famous microscopic study of Delft's canal water and the investigative work of his contempories, Robert Hooke and Athanasius Kercher (who was most likely the first human to witness microscopic life). In these days we had recognised the effects of what we later called microbes but we had little evidence of what was causing them, for who could not wonder what induced a feverish, spluttering epidemic of 'flu? Nor who could not wonder what was controlling the geochemical processes occurring across the Earth?  We just did not have the tools then to probe their world any further in any scientific way.


Will we see Crimean-Congo Hemorrhagic Fever again?

Last Tuesday a man, flying into Glasgow, Scotland from Dubai, was admitted to the local hospital with a very rare disease in these parts - in fact it was the first reported clinical case of the disease here. It is known as Crimean-Congo Hemorrhagic Fever, or CCHF and it subsequently emerged that after being transported to a specialist centre in London he later died at the weekend, a horrible death probably characterised by rapid onset of fever, headaches, hemorrhage, intestinal damage and neurological symptoms.
 
This particular disease is caused by a virus that we know quite little about. The CCHF-virus (CCHFV) and we have nothing in our grasp to stop or prevent it but it is one that we our keeping a very, very close eye on.  Hence the big interest in the recent case in Glasgow. This virus can have a case-fatality rates of upwards of 30%, however the rate of subclinical infections is largely unknown but might be close to 90%.


CCHFV is a very geographically widespread virus found across two major continents and about 30 countries. Thankfully not yet in North-Western Europe and the UK but it is found across much of Eurasia (Eastern Europe, Asia and the Middle East) as well as Africa. It was first seen in the Crimean peninsula during the 1940's and later popped up in the Congo (hence it's name) but don't let that fool you, it's not as restricted as that might let you believe. There is evidence that its range is even increasing.

Now, CCHFV is one of those awkward viruses, it has a rather complicated lifestyle choice. It is zoonotic as in it comes from animals and is not like measles and mumps. In order to survive it has to infect both ticks (a kind of blood-eating invertebrate arachnid) and vertebrates, like domestic animals or even humans. But the major risk factor for us are tick bites. And to complicate matters even further, the young virus-laden tick particularly likes to live out its youth on the backs of smaller vertebrates like hedgehogs and only once it has matured can it jump to cattle, sheep and goats plus some species of birds (the kind of animals that we like to have around us. The virus is a two-host parasite and so is the tick. The virus can even spread from tick to tick during reproduction (sex and birth) and it can even move from vertebrate to vertebrate, given close contact with infected bodily fluids. It is this final property that makes public health workers so worried: it can really kick off around an ill-prepared hospital.

CCHFV is a bunyavirus, like schmallenberg virus.

So to understand why CCHFV is where it is, you have to understand this cycle of infection and given that the same domestic animals are found throughout the world, the major controller of CCHFV presence are the ticks. It is a pretty old and genetically diverse virus which probably spread across its range with the expansion of agriculture from the Middle East across Asia and down in Sub-Sahaharan Africa.

One of the most favourite things of CCHFV are Hyaloma species of ticks, in particularly Hyaloma marginatum. These hard-bodied ticks live for about 3 months and as described above pack that short life full of multiple host changes. These ticks are also pretty widespread across the world but they really prefer dry and open habitats full of their small and large vertebrate hosts. Worryingly these species of tick have been found in areas of South Europe surrounding the mediterranean and has even been found as far north as the UK, carried there by migratory birds. Recently, CCHFV infected ticks were even detected in Spain found on deer.However in both these places, endemic or even epidemic outbreaks of the virus have not been observed. Perhaps the virus is not completing its full life cycle there or maybe infections do occur but are sub-clinical. A recent analysis suggested that the risk to more northerly European regions is low, citing cold springs that would prevent the ticks from surviving. But that doesn't stop Europe from worrying as two years they published a report mentioning that:

 "....a rise in temperature and a decrease in rainfall in the Mediterranean region will result in a sharp increase in the suitable habitat areas for H. marginatum and its expansion towards the north, with the highest impact noted at the margins of its current geographic range"

That's right. One of the major issues with tick borne pathogens is the changing environment, whether it is climate change or agricultural growth. Here's a great review on the role of climate change on the distribution of ticks. One of their points regarding CCHFV is that very little is understood about how this virus interacts with ticks (we don't exactly know what species it infects or how) and their vertebrate hosts (what animals are infected in the wild). So to answer the question at the start: will UK ever see CCHFV again? I wouldnt be suprised if another imported case springs up from endemic regions but whether it will establish itself here is not so predictable. Certainly at the minute it cant: our weather is much too harsh for the tick. But what about in the future as climate change alters the environment in and around the Mediterannean? We're going to need more research into the virus to answer the above questions. CCHFV is an important global pathogen and an unmet medical need.

Deadly viruses, bats and Python Cave

 How would you like to adventure inside a tunnel that is 15 metres in length, 12 metres wide and in parts only 3.5 metres tall? Doesn't sound too bad. The catch is that this very popular tourist attraction - known as 'Python Cave' in Queen Elizabeth National Park in Uganda - is crammed full of 40,000 Egyptian fruit bats, African rock pythons (who actually feed on those bats) and a couple of forest cobras. It's also incredibly dark and piled with bat poo but most importantly it's known to harbour one scary virus: Marburg virus.


This cave has already lead to the death of one Dutch tourist and the infection of one from the US sparking major scientific interest in it. This story is nicely written up in the recent book: Spillover by David Quammen. But up until a couple of years ago we didn't know how these people got the virus and that's why the Centers for Disease Control, alongside teams from South Africa and Uganda itself have made it their mission to uncover the secrets of Python Cave and specifically, why did those tourists get infected at that particular place and time? If we know that, maybe we can rationally defend ourselves against getting infected. Well,  a recent paper appears to answer that question.
 
Publishing in PLoS Pathogens today, the CDC lead team report a detailed ecological investigation into Python Cave and especially the Fruit bats that roost there. In 2009 the same team documented their initial study of a nearby cave associated with an outbreak of marburg virus in miners working there. They found that the Egyptian Fruit bats roosting inside harboured a very diverse mixture of marburg viruses, indicating that they were the natural source of the infections. For an excellent set of pictures of the bats, including a nice shot of a bat and a python, see here: http://www.flickr.com/photos/simian_fan/sets/72157594342657070/.

A schematic of a marburg virus particle - from ViralZone.

So when the next outbreaks happened they repeated the analaysis in that cave to see if the same bats were the source again, involving the capture and analysis of over 1,500 individual bats. But this time they were able to assess how virus infection in the bats differed over their lifetime and from season to season and through this they were able to identify some alarming patterns. They wanted to know precisely whether these bats still contained marburg, how did they transmit the infection to each other and to humans and how did the virus persist among the population?

Using sensitive PCR analysis combined with antibody testing and virus isolation, just less than one fifth of all their bats sampled showed some evidence of current or past infection with marburg virus. And these viruses were pretty similiar to those found in the nearby mine - even one of the bats had been tracked all the way from the mine indicating long range transport of infection. Inside each of the bats they were able to find evidence of the virus in numerous organs, like the lungs, kidney, blood, colon and even their reproductive tract indicating that infection could be transmitted via multiple routes: poo, urine, biting and scratches, through birth and even via ticks and other biting insects.
the peaks and troughs of marbug infection correlate with birthing in the bat colony

But one of the most interesting findings was this: bats of around 6 months of age were the major carriers of the virus. Adults only showed a relatively low and constant level of infection, while very young pups had low levels indicating that virus infection peaks at a particular age level and then the incidence shoots back down. Clearly something is controlling this pattern of infection but what that is is unknown. Yet when they alligned their virus detection data with historical numbers on when humans had been infected with marburg in the past, they showed that the peak in risk of symptomatic infection correlates with the bat birthing season, the time when pups born in the last season reach 6 months of age. They predict that every year a total of 20,000 pups are born. That is a lot of virus infected baby bats. We've even seen this pattern before.

So what the hell is the take home message here? Ok so we know bat populations all around the world contain masses of viruses and in the future we're only going to find a heck of a lot more. And in many cases these viruses have jumped species and killed lots of people and nearby animals and the rate of this may even be increasing. And probably many of our common pathogens nowadays, like mumps and maybe even the common cold originally came from bats. So in the future we would like to stop it happening - lots of lives and money could be saved. 

But it's probably very hard and ethically wrong to a) kill all the bats, b) vaccinate against every bat-borne potential virus and c) do nothing. So what many people have called for is a more general, ecological answer to address the whole swathe of potential emerging pathogens and this paper begins to lay down concrete data that provides one mechanism to prevent virus emergence: don't go anywhere near bat roosts during birthing season if you can help it. Of course these answers will probably also lie in the spheres of cultural and economic changes in behaviour as well. So remember that this study of marburg in Africa is but one peice of a very large and complicated ecological problem.

Amman BR, Carroll SA, Reed ZD, Sealy TK, Balinandi S, et al. (2012) Seasonal Pulses of Marburg Virus Circulation in Juvenile Rousettus aegyptiacus Bats Coincide with Periods of Increased Risk of Human Infection. PLoS Pathog 8(10): e1002877. doi:10.1371/journal.ppat.1002877

A new coronavirus, should you care?

I doubt you have missed the news but a new virus that infects (and has  so far killed one person) has just been discovered in the last few months. The virus in question is believed to be a new - never before seen in the wild - kind of virus (a new coronavirus to be more precise), so we really have little clues as to how it behaves as not much work has been done.

Schematic of a coronavirus - this new virus probably looks a lot like this. From Biowiki. 

We only have two examples of human infections with this new virus to go on but despite this, the BBC and other media outlets have sparked confusion (and maybe panic) by comparing it to the 2002 SARS coronavirus (whose case fatality rate was around 10% of those over eight thousand or so people infected), which proved to be a much more deadly affair. What they probably should have compared it to is the common cold coronavirus, known as 229E - an equally valid example.

But this misses the point, it is all speculation really at this minute in time. We should really wait for the hard facts to emerge. So what do we actually know?

What's happened so far?

We first became aware of this new virus (it doesn't have a name yet - nor is there any published material on it - that's how new it is) a few months back when the Erasmus Medical Centre in the Netherlands discovered the virus in a fatal case of lung disease from a Saudi national. A couple of weeks ago, it was spotted again, this time in a Qatari citizen travelling from Saudi Arabia and Qatar. So far this man has not died (he's in intensive care in the UK) but he was suffering from 'acute respiratory syndrome and renal failure' when he was airlifted to the UK and their Health Protection Agency identified the virus. By sequencing the virus's genome, the UK team confirmed it was highly similar to the Dutch sequenced one. However, this sequence has not yet been published so we don't know how this relates to the hundreds of thousands of other coronaviruses out there.

As you can predict, with such a limited understanding of this virus there are many, many questions about it. These are important questions that ultimately impact on public health and no doubt these will be answered in the coming months.

What is a coronavirus?


These coronaviruses are rather large and encased within a fatty membrane and have a very, very large genome (around 30,000 nucleotides) made up of RNA with positive sense polarity. Encoded within this massive genome are ten genes that produce a lot more proteins due to some viral tricks. These proteins are what allow it to infect and enter cells (in this case human airway cells), replicate and make new virus particles. And of course combat the immune system at every step of the way. The genome of this new virus has yet to be published so we cant comment on how it's genes look and function.

Where did it come from?

We don't know where this virus came from nor why only now are we seeing it. There is also a chance that this virus could have always been in humans but that only due to sensitive lab tests like PCR and deep sequencing we were able to detect it. Although if it does turn out to only cause severe respiratory disease this is probably not the case. Of course the other theory is that this represents the first few infections of this virus into humans, probably emerging from an animal reservoir in the middle east. Sequencing of the virus and comparison of it's genome with other known animal coronaviruses (avian or bat?) may be able to pin point where, when and how it came to infect these two men.

How dangerous is it? 

So far we know of only two cases of this virus infecting humans. N =2 is not much of a sample size to draw any meaningful conclusions. In both men, it is thought this virus caused serious episodes of respiratory disease but without understanding how many other people got infected and who presented with sub-clinical or only mild disease we can't comment on how dangerous it really is.

The HPA are aware of a number of other cases of respiratory disease in the middle east but yet these aren;t confirmed to have have anything to do with this new virus. But so far, preliminary follow up studies on the contacts of these two men have yet to pick up any cases of significant disease despite these two men being well passed the viruses incubation period and peak of infectiousness. The ability to detect whether people have been infected in the past via antibody testing will surely clear this mystery up.

What can we do about it? 

Not much. There's no vaccine or no cure but remember that despite this virus kicking around for at least months/weeks, only two cases have been discovered. But anybody returning from the Middle East should be aware of any respiratory symptoms as should anyone associating with people returning from these countries. The countries in question should also be keeping a close eye on clusters of disease and the origins of the virus. Currently the virus doesn't appear to be very infectious or it is highly infectious but causes little or no detectable symptoms. Both theories would fit in with the fact that we have seen no disease in the two men's contacts.

So is it like SARS or is it more like 229E? Or something entirely different? As is often the case, only time and science will tell so lets focus on the facts and concentrate on doing important epidemiological, genetic and virology work done.


Will microbiologists ever have a Lesula moment?

Last week there was a report in PLoS One documenting the first scientific description (pictures, morphology, behaviour and even genetics) on a newly identified species of primate living in and around the Congo river basin. Its name is a Lesula. And it is magnificent looking.

Haunting picture of an adult Lesula's face. From Hart et al 2012 PLoS ONE
Im sure you've read about it and I'm sure you've seen those amazing pictures. This monkey, a close relative of green and vervet monkeys, had previously been known the local population but only in 2007 when a chance encounter between a field team and a captive member of species did the monkey start it's journey to becoming known to science. What followed was the scientific discovery that lead to this paper.

What is so amazing about the whole story is that it has gone this whole time without being documented. This is only the second new primate to be discovered in Africa in the last 28 years. A newly documented mammalian species is something of a rarity these days but this got me thinking: what does this have to tell us about the future of microbe discovery and our understanding of microbial diversity out there?

It's a long shot of a thought but it really did start me thinking.

Modern microbiology with its focus on metagenomics and deep sequencing alongside targeted microbe discovery projects like those looking into the kinds of viruses bats harbor is paralleling what happened with animal and plant taxonomy when western scientists first began exploring and documenting the living world around them. Only back then we didn't really know what bacteria or viruses were let alone did we have the technology to accurately study them. It's not surprising that we were only aware of a handful of microbes. But we are catching up now. Nearly every week a paper comes into my email inbox telling me of the discovery of another new virus or at least viral sequences that act as a clue that a virus-like thing was here.

I wonder how long it will take us to reach our Lesula moment? Will we ever see it? What will microbiology look like if we do? Will we ever say: "Oh, this amazing! Nobody has found a new virus or bacteria in the last 30 years!" ?




Neil Gaiman: "Make good art"


Watch this video. Watch it now. It goes alongside that of the late Steve Jobs talking at Stanford but this time it is Neil Gaiman. And this time it is at Philadelphia University of Arts in 2012. Listen to the author build upon the motto: "Make good art". He fills the 19 minutes with dozens of worthwhile, distilled quanta of advice for young journalists and writers. But where is the talk: "Make good science"? Where are the role models for young researchers? Why are they not being recorded giving commencement addresses on doing good science? In the meantime until they step forward, Neil's advice may be quite easily transferred to the fields of science.

A bat virus that can't fight your immune system

I have been too busy to go into this in much detail but I'm wondering whether anyone had any ideas as to why this virus (cedar virus - a newly discovered bat RNA virus) appears not to be able to combat the human innate immune system, specifically: HeLa cell secretion of beta interferon in cell culture.

 1) is this real?

2) does a virus have to be able to combat this part of the immune system?

3) why use a human  cell culture to study a bat virus? and finally,

 4) what does this mean for the virus in a bat?


Cedar Virus: A Novel Henipavirus Isolated from Australian Bats



Abstract:

The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin- B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-╬▓ response than HeV.

Schmallenberg virus hits Europe again but should we be worried?

Lambs, don't worry. I's not all that bad.

I wrote back in February this year (2012) of a worrying outbreak of disease spreading across North-Western Europe, starting in Germany and ending up in the UK and even Spain and Italy.

The disease, manifesting itself in the Spring lambing/calving season, as fetal deformaties in these ruminants (also now alpacas). Many of which lead to the death of the newborn animal. It was quickly uncovered that this illness was caused by a previously unheard-of virus (an orthobunyavirus if you wanna know) now named after the town it was sampled from: Schmallenberg.

To date, no cases of human infection have been reported. And currently, cases have severely decreased as we leaving lambing/calving season.

Back seven months ago we really didn't know an awful lot about this emerging virus apart from that it looked not much like anything we had seen before. We didn't know how it got to Europe, we didn't know if it was going to stay here and we didn't know what to do about it. It was predicted that the virus was spread by midge flies living across Europe during the summer of 2011, the animals exhibiting a flu-like illness that barely registered with farmers. But when it came to the spring and the then newly pregnant sheep or cows gave birth, it soon became evident what damage the virus had done in this group of individuals.

What has happened since the Spring?

The months following this initial characterisation was a worrying one but scientifically really interesting:

I wrote in May that we had discovered where the virus had come from. A Japanese group had sequenced a load of new viruses that were cousins on Schmallenberg. One of the reasons we didn't know where the virus originated was due to really not knowing much about the genetic diversity of the orthobunyaviruses.

Bunyavirus - segmented genome. Is Schmallenberg composed of  segments from different viruses?

They then compared their sequences with the German isolate from back in early 2010. It became quickly obvious that parts of the viruses segmented genome came from different viruses and was hence a 'recombinant'. Recombining - or joining - together 3 fragments from two different kinds of viruses. But now with further increased sampling of orthobunyavirus genomes, Schmallenberg appears to be firmly rooted in one particular viral group while another virus cousin seems to be a recombinant.

A couple of months later, a Danish group showed that they could detect Schmallenberg virus RNA in one particular group of midges: Cullicoides obsoletos in Autumn 2011, when the virus was spreading across Europe, including Denmark.  

Culicoides sp. Reported vector for Schmallenberg virus.

Europe's stance downgraded but is it still here?

But Defra reported in June that the lack of seriousness of Schmallenberg virus had caused them to downgrade their view on the disease after only 0.002% of the susceptible ruminant population was affected. And those that were infected caused little negative impact to the farming.
SBV is no longer considered an emerging disease and therefore affected Member
States will no longer be reporting to the OIE on a regular basis. It was agreed at the
OIE 80th General Session that the disease is low impact with no public health risk
and negligible risk posed by commodities such as meat, milk, semen and embryos.
Thankfully European Food Safety Authority (EFSA) earmarked a 3 millions Euros for researchers to look into the biology of how Schmallenberg virus infects and causes disease in animals. Although nothing ear-marked for vaccine research. But despite this downgrading, the virus is in the news again when scientists at the UK Institute for Animal Health report (although there's no paper available) that they are still seeing new evidence of infection in cows in one of their farms when they look for antibodies.  The evidence has not been repeated in mainland Europe.

The UK guys take this as evidence that the virus survived the winter in the UK midges and is beginning to spread across the country. Although I'm not sure how much this could be explained by some animals mounting a slower response to the virus from the season before that. But maybe a year is too long for this.

Depending on midge dynamics across the country, which is itself dependent on weather and temperature etc (midges don't like the cold and the rain). The extent of the virus spreading and infecting new animals may be blocked by some animals having already mounted an immune response against it from last year and could be protected. You could  predict that this could drive the virus into areas that were not affected a year ago.

OK - it might be here, but calm down.

But as Defra and the EFSA stress, this virus poses no risk to humans and has only - to date - had a small  effect on European farming industry. It is no bluetongue disease however, I'm sure it's distressing for the individual farmers and it could have a relatively large effect on single farms that have been heavily affected. But in general,  even if this virus does return this summer (and we see its effects next Spring) hindsight has shown us that we shouldn't be as worried as we were 6 month ago.

Who cares about ebola?

Allow me to play the Devil's advocate:

Ebola has struck again in Uganda, and to date, at least 15 people have died from it and a further 32 are in isolation in the country. This is the most recent outbreak in what appears to be an annual occurrence in Central Africa. What I'd like to know is: why do we care?

Numbers of African outbreaks since 1976 to 2012, based on CDC records. 

The African strains at least, of Ebola are probably not as fatal in humans as reported (see Vincent Racaniellos take here), as a disease it doesn't really effect all that many people and I think Africa has a lot more pressing public health issues than really worrying about a virus like this one - malaria, vaccination campaigns and malnutrition.

The ebola belt traces the area of rain/deciduous forest covering Central Africa from the Cote d'Ivore in the West to Uganda and South Sudan in the East. Somewhere inside that forest lies in wait, Ebola. 

Currently a lot of research is being carried out on Ebola and related viruses and we are making excellent progress in understanding how the virus infects and replicates in our cells but also how our body responds to it and how this leads to the fatal, often haemorrhagic, fever that is ebola.We are even beginning to understand how this virus, a virus of fruit bats, can spill over into other animal populations nearby, like primates and us. 

All this information could help lead to the development of antiviral molecules or even an ebola vaccine or even a potential preventative measure based on some sort of control of host species.

But are there more useful things for us to spend our money on? Which of the above areas of research would be the most cost effective and successful? Is research on ebola pursued on the back of a potential bioterrorism concern rather than on public health thinking?

Why should we care about ebola? Answers on a postcare below.

No shit - a new way to study diarrhoeal disease

Diarrhoeal disease is awful. I don't think I have to tell anyone that. 

According to the WHO:

Key facts
  • Diarrhoeal disease is the second leading cause of death in children under five years old. It is both preventable and treatable.
  • Diarrhoeal disease kills 1.5 million children every year.
  • Globally, there are about two billion cases of diarrhoeal disease every year.
  • Diarrhoeal disease mainly affects children under two years old.
  • Diarrhoea is a leading cause of malnutrition in children under five years old.

Behold the wonder of the intestine
 The major causes of disease are infectious agents, namely the likes of noroviruses or rotaviruses. (very good wikipedia article here) But also bacteria, parasites and non microbial assaults. These pathogens enter our bodies via the oro-faecal route where they are able to infect the lining of our intestinal tract. Here they exert their biological effects and manipulate their resident tissue to aid in their replication and spread in the general population. In areas with poor sanitation this is why these diseases are such massive killers. They are acutely adapted to this way of life.

We are somewhat out of our grasp when dealing with - and studying - these organisms. In many cases (noroviruses especially) we cannot even grow the viruses in the lab. Many groups use mouse noroviruses and immunocompromised mice but of course this really isn't optimal for human viruses - especially when pathogenesis and drug development/vaccine studies come along.

So, if we cannot grow them how can we study them? Plus even if we did have the ability to culture them we lack accurate model systems of the human gut to even discover anything worthwhile about the way they grow.

But imagine if we had the ability to study human viruses in the lab, which had been themselves grown in the lab, with human tissue which had also been grown in the lab. 


Enter the human intestinal organoid.  

An intestinal organoid a la Spence et al 2011


An organoid is a structure that resembles an organ. It is not an 'organ' itself taken from a body - it's constructed in the lab to function as one. But organs are pretty complex and none less so than the gut. Its complexity resides in the physical (three dimensional) and biological (cell type/gene expression) planes. So how can we build this 'awesome' work of evolutionary engineering ourselves?

Easy.

You just copy mother nature.

Briefly, the NIH-approved embryonic stem cell line WA09 (originating from the WiCell Research Institute and obtained from the Baylor College of Medicine Human Embryonic Stem Cell Core) was cultured using feeder-free conditions. Stem cells were split at a high density, and, once they reached 85 to 90% confluence, cells were treated for 3 days with a series of differentiation media containing activin A to begin differentiation into definitive endoderm. Definitive endoderm was then treated for 2 to 5 days with growth factors Wnt3a and FGF4, leading to formation of hindgut spheroids. Once spheroids spontaneously detached from monolayers, they were collected, embedded into matrigel (BD Biosciences), and supplied with media supplemented with intestinal growth factors (Wnt3a, R-Spondin1, Noggin, and epidermal growth factor [EGF]; all supplied from R&D Systems). Spheroids matured into intestinal organoids over the course of ~1 to 2 months before they were used for experiments.
It started off like this


Anded up like this
Grown in this way you can 'easily' generate what effectively looks and feels like a human intestinal epithelium (these things also contain some underlying mesenhcymal tissue). They can even be kept alive for over 3 months and the stem cells from which they derive can be frozen and re-animated any time to set up more and more organoid cultures.  


So do these organoids allow viral replication? And can we use them to understand how these viruses infect and cause disease? Well the short answer is yes.


A U.S group from Baylor College of Medicine in Texas were able to cut open the spherical structures and infect them with rotaviruses, even clinical isolates of the virus (OA paper here). In this case the virus needed access to the inside of the structure, the part the resembles in insides of our gut and the place where the cell it likes to replicate in are found. These viruses bound to cells, got in and began replicating and generating new viral particles which could go on and initiate a whole new round of growth. This a whole lot better than using primary monkey kidney cells to isolate and grow the virus or using lab adapted strains.


Organoid structures could be infected with rotaviruses


This was only a preliminary observational study showing a proof of concept that this technique which had previously only been done using mouse stem cells, could work for humans and that they could be used for infection studies. What they didn't show was that rotavirus infection of the organoids bore any resemblance pathogenically to infection in humans. I'm sure this is the next step. What also would be of great use would be to see whether other non-culturable but important human pathogens could be grown this way - I'm thinking noroviruses. What is neat about this work is that we have the ability through recombinant gene vectors to knockdown or over-express any gene we want. The initial paper documented this in 2011. And this is being done with an NIH-approved stem cell line - imagine what could be done with healthy or 'diseased' stem cells.

One problem with this lies in the way they were infected. In order to access the inside of the spherical structure the organoid was cut open using a tungsten needle - an extremely sharp instrument. Who know what kind of effect this would have on nearby cells? But I guess without it this work could not be done and this was probably the safest way of doing so.


Another issue is - like the majority of other in vitro models - there is not immune system component to the structure. And of course the innate immune cells residing in tissues would have a wide ranging effect on the subsequent spread and infection kinetics of the virus. However, you could imagine that in the future it may not be too difficult to add these in to the system. 



ResearchBlogging.orgStacy R. Finkbeinera,, Xi-Lei Zenga,, Budi Utamaa,, Robert L. Atmara,b,, Noah F. Shroyerc,, & and Mary K. Estesa,b (2012). Stem Cell-Derived Human Intestinal Organoids as an Infection Model for Rotaviruses mBio, 3 (4) DOI: 10.1128/mBio.00159-12

#microtwjc 5(?) Microbiology of the built environment - the toilet edition



This weeks microbiology twitter journal club is:


Microbial Biogeography of Public Restroom Surfaces


Gilberto E. Flores1, Scott T. Bates1, Dan Knights2, Christian L. Lauber1, Jesse Stombaugh3, Rob Knight3,4, Noah Fierer1,5*
1 Cooperative Institute for Research in Environmental Science, University of Colorado, Boulder, Colorado, United States of America, 2 Department of Computer Science, University of Colorado, Boulder, Colorado, United States of America, 3Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, United States of America, 4 Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, United States of America, 5 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado, United States of America

have a look at the abstract here (emphasis my own):
We spend the majority of our lives indoors where we are constantly exposed to bacteria residing on surfaces. However, the diversity of these surface-associated communities is largely unknown. We explored the biogeographical patterns exhibited by bacteria across ten surfaces within each of twelve public restrooms. Using high-throughput barcoded pyrosequencing of the 16 S rRNA gene, we identified 19 bacterial phyla across all surfaces. Most sequences belonged to four phyla: Actinobacteria,BacteriodetesFirmicutes and Proteobacteria. The communities clustered into three general categories: those found on surfaces associated with toilets, those on the restroom floor, and those found on surfaces routinely touched with hands. On toilet surfaces, gut-associated taxa were more prevalent, suggesting fecal contamination of these surfaces. Floor surfaces were the most diverse of all communities and contained several taxa commonly found in soils. Skin-associated bacteria, especially the Propionibacteriaceae, dominated surfaces routinely touched with our hands. Certain taxa were more common in female than in male restrooms as vagina-associated Lactobacillaceae were widely distributed in female restrooms, likely from urine contamination. Use of the SourceTracker algorithm confirmed many of our taxonomic observations as human skin was the primary source of bacteria on restroom surfaces. Overall, these results demonstrate that restroom surfaces host relatively diverse microbial communities dominated by human-associated bacteria with clear linkages between communities on or in different body sites and those communities found on restroom surfaces. More generally, this work is relevant to the public health field as we show that human-associated microbes are commonly found on restroom surfaces suggesting that bacterial pathogens could readily be transmitted between individuals by the touching of surfaces. Furthermore, we demonstrate that we can use high-throughput analyses of bacterial communities to determine sources of bacteria on indoor surfaces, an approach which could be used to track pathogen transmission and test the efficacy of hygiene practices

Here's my thoughts of this (briefly) 

As you can see from this paper certain American restrooms harbour a massive diversity of bacterial species (only bacteria were looked at - I wonder what kind of viruses are here). The majority of which have a strong association with the human species; others come from soil or water. Most of all the diversity of bacteria in the restrooms come from our skin (no surprise there really). 

You might ask: "So what?", "Who cares?" or "Why bother?" 

Is it just because you had access to some fancy machine and 12 bathrooms?

I have to disagree with this viewpoint: This is only the beginning.

 In order to fully understand the microbiology of the built environment and how it effects the human population residing in it we have to start somewhere - and deep sequencing or bacterial diversity is a damn good place t start. (after all we spend the majority of our time in doors - and even when we're outside it will probably be in or in close proximity to major population centres like cities). 

If you don't believe have a look at this site.

This falls into a larger investigation looking into the microbial diversity found across different environments, such as the office (this groups follow on paper) or nursery. We can study 'healthy' environments and we can study 'unhealthy' environments. Potentially there are important microbiological differences between the two. By determining the baseline microbiome we could in theory begin to specifically alter its appearance to enhance the human experience of the build environment for health, environmental or economic reasons. 

What's the difference between one kid with a fever and one without?

A paper out last month goes a little way to answering a question I have had for a while: what are the major causes of fevers in children?

Imagine this: your child goes to the doctor, they have a fever so they might get given antibiotics or they might be sent home for bed rest because of a non-descript  'viral' infection. But what is that non-descript virus? and how is it causing feverish disease? Maybe if we knew exactly what was doing this we may have some chance to prevent it.


Fevers are an annoyance and in some cases can even be life threatening. Often times they are your bodies way of helping clear an infection. Your body will 'see' and detect infectious (and potentially dangerous) micro-organisms like viruses or bacteria and set off a chain reaction of chemical signals resulting in a rising temperature. But fevers may also be caused by a range of other ailments: arthritis, cancer or basically anything that causes inflammation. 


This paper in PLoS ONE suggests that we should delve into those children's viromes to see what is making them sick. Using next generation sequencing of nasopharyngeal swabs as well as plasma blood samples from febrile and non-febrile American children, they attempt to tease apart they causes of their illness. They also looked with PCR to detect viruses more specifically. This allowed them to essentially cover all viral bases in a comprehensive manner and hopefully detect what was truly in there. 


However, this approach is only based on looking for viral genetic sequences and no search for the actual virus was carried out. This means that those viruses detected could may never have infected the person, could have come directly from the environment or had been recently cleared by the immune system. WIthout virus isolation we cannot be sure to what extent a viral sequence is associated with a certain condition.
The viruses they found by PCR and deep sequencing.

In some cases this knee-jerk reaction to infection actually can damage you and can even result in seizure and death. Diagnosing the cause of fevers and treating early in thus a major concern and of the major issues of fevers is that they can be set off by a multitude of pathogens, the majority of which we have never really studied in much detail. Hence this group spending their time and money investigating it. 


Abstract:

Why did they do the work:

Unexplained fever (UF) is a common problem in children under 3 years old. Although virus infection is suspected to be the cause of most of these fevers, a comprehensive analysis of viruses in samples from children with fever and healthy controls is important for establishing a relationship between viruses and UF.


Methods:


We used unbiased, deep sequencing to analyze 176 nasopharyngeal swabs (NP) and plasma samples from children with UF and afebrile controls, generating an average of 4.6 million sequences per sample. 


Comparing viruses seen in febrile versus afebrile kids. Spot the differences?
Results:


An analysis pipeline was developed to detect viral sequences, which resulted in the identification of sequences from 25 viral genera. These genera included expected pathogens, such as adenoviruses, enteroviruses, and roseoloviruses, plus viruses with unknown pathogenicity. Viruses that were unexpected in NP and plasma samples, such as the astrovirus MLB-2, were also detected. Sequencing allowed identification of virus subtype for some viruses, including roseoloviruses. Highly sensitive PCR assays detected low levels of viruses that were not detected in approximately 5 million sequences, but greater sequencing depth improved sensitivity. On average NP and plasma samples from febrile children contained 1.5- to 5-fold more viral sequences, respectively, than samples from afebrile children. Samples from febrile children contained a broader range of viral genera and contained multiple viral genera more frequently than samples from children without fever. Differences between febrile and afebrile groups were most striking in the plasma samples, where detection of viral sequence may be associated with a disseminated infection. 


Conclusions:


These data indicate that virus infection is associated with UF. Further studies are important in order to establish the range of viral pathogens associated with fever and to understand of the role of viral infection in fever. Ultimately these studies may improve the medical treatment of children with UF by helping avoid antibiotic therapy for children with viral infections.

Points to consider:

As I said previously, the group only looked at viral sequences. No viruses were isolated or grown from these samples which makes it a little complicated in drawing conclusions based on this data. But it is extremely interesting and worthwhile and perhaps in some instances may give us a less biased view into the virome.

We all harbour viral sequences. Even the 'healthy' control populations when you look at their upper respiratory tract and circulation, indicating that the issue of fever versus no fever may be more complicated than expected. Maybe some people are much easily able to withstand disease progression from an infection.

They only looked at plasma and at the upper respiratory tract. Many other parts of the body could be sampled in the future to more comprehensively investigate how the virome of a particular is associated with any particular condition or disease. The digestive tract, urogenital tract or blood cells (not plasma) could and should be investigated.

Causation cannot be determined from this study. And they even say that this was not the aim of the investigation. To determine causation that a particular virus caused fever in kids one would have to fulfil Koch's postulates.

Fever could have been associated with bacterial exposure/infection yet this was not looked at here. Perhaps this may have been more obvious to the diagnosing clinician and antibiotics would have prescribed.

In conclusion, this is an interesting paper but it is in a sense quite preliminary. They have defined their methods and proven they can detect a wide range of viral sequences in clinical samples. But at the minute it is rather difficult to conclude anything else from this. I look forward to further characterisation of the viromes of multiple other tissue samples and even the characterisation of the viruses they identified. Perhaps they will represent entirely unknown disease causing viruses.



Kristine M. Wylie1*, Kathie A. Mihindukulasuriya1, Erica Sodergren1, George M. Weinstock1, Gregory A. Storch2 (2012). Sequence Analysis of the Human Virome in Febrile and Afebrile Children PLoS ONE, 7 (6) : 10.1371/journal.pone.0027735