Harnessing innate immunity to cure HIV

Using a single drug derivative of vitamin A, researchers are now beginning to harness our innate immune system in order to force it to recognise, find and kill any cells infected by the human immunodeficiency virus (HIV), giving hope of a cure in the future. 

HIV particles (yellow) on the surface of a T cell, NIAID/NIH

Response To Infection

The Rule of 6ix blog is back. At least temporarily until I can find another outlet. Recently I have had a lot of science questions and ideas bouncing around my head as I try to define the future of my research and I need to solidify them. Hence the blog. So for the next few weeks or months you can some musings on virus/host interactions here. 

One of the questions I have is:

Why do our bodies’ know we have an infection? 

It might be obvious to you but our bodies are a valued commodity, excellent real estate, a resource worth having. We are composed of trillions of cells; we take in nutrients, excrete toxins and we have all the machinery required to sustain life. Which is excellent. But all this puts us in the line of sight of other bodies looking to use our success and many of them don’t care as much about our well-being as we do.

We inhabit this world with millions of other species: whales, dogs, magpies, frogs are all obvious examples but by far the most common are microscopic life-forms (the ones we can't see without a microscope), such as bacteria, Achaea, single-celled eukaryotes, like yeast, and viruses. These other kinds of life are just as evolved and sophisticated as ourselves, yet some species of microbes have turned to a parasitic way of making a living and physically associate themselves with us, our bodies’ and our cells, in order to survive. This association is commonly referred to as ‘infection’.

Viruses: extreme parasites

At the extreme end of parasitism are the viruses. In general terms viruses are genetic material encased in a protein shell that can get inside cells. The genome provides all the instructions to make new viruses and infect new cells and thus spread from cell to cell and individual to individual. However, these microscopic agents are obligate parasites in that there is no example of a free-living virus (this is because viruses do not encode the machinery required for translation of messenger RNA molecules, the ribosome, probably because of its sheer size). Thus we are essential for their very existence and in most instances this interaction is one-sided and we do not need it for our survival. And it comes at a cost to us.

Viruses (and other parasites) remove resources from us that we would have used for some other task, such as repairing a cell, making crucial enzymes or communicating between cells. Of course viruses can also physically damage our cells as well. But in general all of this is bad news. 

From detection to response

Thus cells and organisms have evolved means to tell when they are being parasitized upon. Yet what’s the point in being able to tell if you have been infected without being able to do something about it? Thus we are able to couple detection with response and it is this response that is tasked with stopping an infection. And a response that arises following infection is thought of as an immune response. Over evolutionary time, those organisms that have defences against infection have prospered because infection is bad for our long-term survival. 

However, there is a dark side to our immune response. Sometimes virus-associated disease is caused by our own response to infection. Our immune response can be damaging. Viruses are so intimately associated with us that we get hurt a bit when we are trying to rid ourselves from infection. Viruses live inside cells and therefore if we want to remove the virus you may have to remove your own cells.  In many cases we have achieved a balance with our viruses. However, sometimes this balance can be tipped in favour of the host or the parasite. Either the parasite is cleared before it has the chance to spread or the host responds in such a way that it irreversibly damages itself. 

Questions, questions and questions

It should be obvious now to you WHY our bodies can tell we are infected but from this idea stems many, many more questions, ideas and areas of future research. Questions like:

1) How do we physically detect infectious agents? What do we detect? What makes them different to us? Do they have to be 'infectious'?

2) How do we respond? What kinds of changes are made?What's responsible for stopping an infection?

3) How do our parasites influence our response? 

4) How do we regulate the response? Especially if it can be damaging. 

5) How important is this response to health and disease? 

6) Why do some individuals respond differently to the same infection?

7) Why do different infectious agents give different responses? And what are these responses?

8) How has this interaction affected the evolutionary trajectories of host and infectious agent?

9) How can we manipulate this interaction? Can we use this knowledge to make better, safer drugs and vaccines?

10) How can we study this whole phenomenon

 experimentally?

I hope to explore these questions in future writing and research.

Rule of 6ix has moved

If you are looking for more virological-related blogging head over to postdocinvirology as Rule of 6ix is no more. I have decided to slightly alter my blogging outlet to reflect the evolution of my working life. I have recently taken up a postdoc position at the MRC Centre for Virus Research in Glasgow in the lab of John McLauchlan. No more negative sense RNA viruses, no more Belfast. This new position focuses on hepatitis C virus, a very different virus (kind of) from what I am used to. And seeming that the name rule of 6ix came from a phenomenon observed in paramyxoviruses (see here on background) I have decided to discontinue this blogging 'brand' in favour of a new up-to-date one. Hopefully the output wont change significantly. But do expect more hepatitis C....

Do carbohydrates play a role In intrinsic immunity?

A question that has been on my mind for the last few months is this: how do viruses interact with cellular carbohydrates? You may find that a dull question but excuse me, I'm currently writing up my Ph.D and these kinds of questions can plague your mind. 

Some example polysaccharide complexity (bioweb.wku.edu)

We all know about the role of proteins, DNA and RNA and lipids in cell biology (we all learnt about this school), but what about carbohydrates, or sugars? Apart from their use in your cell metabolism, what else are they doing? Do they have a structural role? Do they do anything in the immune system? And, if so, how do pathogens circumvent this? Or how does your own cells manipulate them to prevent infection?

Some basic carbohydrate chemistry

As I said, apart from being used during respiration and as an energy store, they can be stuck on to proteins and lipids (glycoproteins, glycolipids and proteoglycans), which can then can be moulded into large, complex macromolecular structures through the use of glycosidic bonding (covalent bonds involving at least one sugar). This is similar to what is seen with amino acids and nucleotides when building polypeptides and DNA, for example. To give an example of the potential for complexity in these large sugar macromolecules: single carbohydrates (monosaccharides) can be joined together to form di- and tri-saccharides, which can in turn be sculpted into larger polysaccharide structures. With each new saccharide added comes an ever increasing number of potential new carbohydrate linkages. With each new linkage, different kinds of saccharides can be attached. And so, and so on. You can imagine what kinds of structures can be assembled using this chemistry. But for what use?

A role in immunity?

This is a question I've been thinking a lot about lately (as I said, finishing up my PhD and part of the work involved viral interactions with sugars). From my limited experience with this is that all I see are viruses using these sugars to latch onto and infect a cell. Think of how influenza uses sialic acids ( a type of sugar). These virus entry receptors, as they're called, are found on the surface of cells. But I can't imagine that these molecules are only being used for the good of our viral parasites. So what is their natural role?

Well these molecules have lots of different roles but one of their more interesting roles (my opinion) is in assembly of what is known as the cell 'glycocalyx'. I like to think of it as a sugary coat of armour. This is a layer of sugar coated proteins and lipids found on the surface of all our cells, forming a lattice-like structure where it can be easily used to alter cell-to-cell interactions, depending on its chemical composition, which as I mentioned earlier is a pretty complex affair. Not easy to go into in a blog post. One emerging function of the glycocalyx is a protective, intrinsic immunological role. It is in a perfect position to physically sift out incoming pathogens that would would like to infect our cells. Especially considering its size in comparison to some human pathogens (around 50 nm - 500 nm). It is also highly sialylated (terminally linked to sialic acids) and sulphated, making it very negatively charged. Viral membranes are also  It is a difficult barrier to traverse. For more information on virus/glycocalyx interactions see this book chapter here. And if you really want to dive into it more, see this great science consortium website (lots of free, open data).

The best picture of the glycocalyx, this time from an endothelial cell (Hubrecht.edu)

Show me the evidence. 

The paper that alerted me to this phenomenon was this: 

Glycocalyx restricts adenoviral vector access to apical receptors expressed on respiratory epithelium in vitro and in vivo: role for tethered mucins as barriers to lumenal infection.

by Stonebraker et al in the Pickles lab. 

This paper, from about ten years ago, basically describes an attempt to get a genetically modified adenovirus into differentiated human airway cells in cell culture. They find that despite expressing the viral receptor on the surface of each cell (and on the cilia), this virus still fails to infect. Something is therefore blocking infection. (Whether there is something biologically wrong with the receptor being expressed in this non natural way (it is usually found on the bottom of the cell) is unknown. Despite any of these concerns the removal of certain sugar molecules and proteins from the surface of cells allows a more efficient infection process to occur. They conclude from this that the cell glycocalyx functions to prevent virus infection. They cannot say why this occurs but postulate that it could physically bind virus particles or may physically sift them out and prevent them reaching the cell membrane where its receptor is found. The paper itself isn't clear cut but these data are presented nicely. Despite being a bit of an artificially system it makes me wonder how other viruses navigate this glycocalyx barrier? Especially for viruses that use sugars as receptors, how do they avoid binding to non-receptor molecules not on the cell membrane? 

For example, consider influenza. It is a virus covered in proteins that like to bind to certain sialic acids on the end of sugar chains at the surface of the cell. It uses this binding to stimulate cell endocytosis and fusion if it's membrane with the cells. How do you suppose an influenza virus particle navigates the glycocalyx? I really don't think we know this answer, even after then ten years since this paper was published. But I imagine that with the work of the Functional Glycomics Consortium we are going to begin to understand this a lot better. 

We should continue to study camels if we care about zoonotic diseases

Dromedarian camel

Inspired by the latest evidence suggesting that camels may have been infected with a close relative of the MERS-CoV (read this from CIDRAP for a nice coverage of the paper) , here's a few top camel facts regarding their relationship with viruses, other wildlife and humans.  Draw your own conclusions from these points.

1) They are found on three continents (Africa, Eurasia and Australia - only here for 150 years) and used to be found on many more (North/South America) but were driven to extinction there probably by climate change or by the actions of humans. Their current wide distribution across the world would allow contact with many other wild (or domestic) animals, and of course this means their viruses. One of the species that camels would undoubtable interact with are humans, but that's because we domesticated them.

They are also found on the Canary islands (brought their by the Moors). Here they are the most important livestock animal and are heavily adapted to that particular environment. This population has been shut off from the other camel populations in Africa for 20 years, so just how is the positive  antibody results explained? Even the canary island bats are unique.

2) Thousands of years (3-6)  ago humans domesticated the camel (Romans used them for military uses). They have been domesticated by humans for food (milk and meat) and work. Oh, and fun. The only wild camel populations left are left are in the Gobi desert. There are feral herds in Australia however. You can milk a camel and get milk and then make cheese or yoghurt. You can see why they would be so popular. Think of them as movable larders. Also, a dead camel is a lot of meat as well and roasted camel has been used for feasts. They are also considered very high value ($250,000 each) and thus their potential worth would probably prevent common slaughtering of the animals.

They are deemed unclean by Islam (however you can easily circumvent this law) and Judaism.

3) They can live until they are 50 - that's a long time if you consider how many infections you pick up over a year. So interpreting antibody evidence may not tell you much. Could this explain the antibodies in Canary island camels?

4) Camel antibodies (single chain 'nanobodies') have interesting properties when compared to human antibodies. This could potentially have implications for immunity to viral infections if you consider how potent they are, their half life or their tissue permeability and hence access to barrier sites.

5) There are a lot of camels out there. Over 14 million to be precise. They show an uneven distribution with the highest concentration is in the horn of Africa, a hot spot of biological diversity, economic disparity and a major trade hub (including with Saudi Arabia - in the past this exchange has already lead to Rift Valley fever epidemics.) . "On an average day, 300-400 heads of goats and sheep, 120-150 heads of cattle and about 50-60 camels are sold," Farah said."  This album speaks volumes.  The kind of place an emerging virus thrives in. Two pictures in this Flickr album highlights why you should watch this area. 1 and 2. This is even more interesting with the discovery of diverse coronaviruses in African bats.

" Cash is received at town markets for male camels sold for slaughter at the age of six to seven years. They are collected at regular intervals into large herds and driven to the meat markets in Egypt, where they bring in profits ranging from LSd 7 000 to LSd 12 000, i.e. approximately US$600 to US$1000, per head. "from wikipedia.

6) They get disease (unlike what we thinks happens with bats) from infections and can pass these on to humans and other animals. There has even been an example of camel to human plague transmission. 

It looks to me that camels are in a fairly rare position of being so numerous and found across so much of the world that they could easily have picked up any number of potentially zoonotic viruses. Even these population densities could even support endemic transmission of camel-adapted viruses. Until MERS-CoV is isolated from camels and shown to be maintained in the populations we should keep this theory in mind that MERS could be a camel-specific virus. The close contact between humans and camels also would facilitate a relative fluid movement of microbes and thus camels could easily act as a amplification host for these kinds of zoonotic viruses. What is worrying is that camel trade and movements between diverse ecologies (tropical central/East Africa and the Middle East) via unique trading hubs could rapidly sample and spread a large swathe of microbial diversity out there, from bats, other mammals or birds. Or even from humans in a reverse zoonosis kind of way. I feel that this antibody work is only a small piece in the MERS puzzle and that camels may act as a virus indicator species in this area of world. If you study camels intensely you may find more viral surprises and could rapidly inform public health policy.

European Influenza virus characterisation published - we're winning for now.

It always amazes me how much effort that our society goes to to track infectious diseases as they spread across the world. Really this probably shouldn't be so surprising to me considering the worry, morbidity and mortality that these infections leave in their wake. One of these infections is of course, influenza and today the European Centres for Disease Control (ECDC) published their characterisation of this seasons influenza viruses. You can read it here and it's worth it, it really is. http://ecdc.europa.eu/en/publications/Publications/Forms/ECDC_DispForm.aspx?ID=1180

Two things are striking in it: 1) Influenza is continuously changing its genome and evolving as well as the relative proportions of each sub-type, as evidenced by sequencing and amino acid sequence production. 

2) Looks like these viruses haven't YET evolved in a way that renders this season's vaccine non-functional. So this season we are winning, at least for now.

So remember that doing PCRs, growing viruses and sequencing them doesn't have to be boring and useless....

Negative Strand Virus meeting 2013

I have just returned from the Negative Strand Virus meeting held in the city of Granada in the south of Spain. The above is the view from the historic Alhambra palace overlooking the city. This 6 day conference, which usually takes place every three years, is the largest congress of students and professional scientists who investigate the biology of those viruses that utilise a negative sense RNA genome to survive, whether they are single stranded like measles or rabies or segmented like influenza and the bunyaviruses. The event focuses on entry, structural biology, replication, pathogenesis and fighting viruses with antiviral s and vaccines. There is also a considerable emphasis on how viruses evolve. 

For me, the meeting was a real success, getting to hear many of the eminent virologists speaking discuss their findings. It was also tremendous fun thinking about all their recent work and meeting many of them in person. At this meeting I presented a poster outlining my own investigations and was able to discuss the meaning of my work with the conference delegates. 

All in all it was a great meeting and lots of fun outside of the conference itself. Hopefully I met some people which I will run into again and again in the future. 

Naming a viral disease around the world

OK so I need a little help.

I am a final year PhD student studying the molecular biology of mumps virus. As part of my final written thesis I would like to include an historical aside to mumps virus and the disease. In particular I would like to know how different societies have named the same set of characteristic symptoms: swollen salivary glands and testis. (Of course there are others, such as meningitis and many infections are asymptotic but lets not be confusing.)

For this I need diverse and global input from as many people as possible. So if you have a name in mind, add it to this spreadsheet.

I have filled it in with a couple of known names to me.

Mumps - characteristic unilateral salivary gland swelling. What do you know this as?

UPDATED: 10 things we need to find out about the #NCoV

Following a conversation on twitter on NCoV-EMC, I quickly realised that I did not know enough about this virus. But then I realised that it is that NOBODY knows a lot about it. There are very little answers to a growing list of questions (for whatever politically/funding/technical reasons).

So, here are a few questions that I think really need answered about the novel emerging coronavirus (I.E if you gave me infinite amounts of money, PhD students and post-docs this is what I would look at). If you have thoughts on them (think they're rubbish/not important/drastically important) or have ways to answer them, please comment below!

1) Is the NCoV-EMC isolated the sole causative agent of the viral pneumonia observed across the Arabian Peninsula and Europe?

2) How many humans have been exposed/infected?

3) What is the true case fatality rate?

4) What is/are the reservoir specie(s)?

5) What animal species have been exposed/infected?

6)Why has it emerged/only been detected in the last year?

7)  How efficient is human-human transmission?

8) How does NCoV-EMC induce disease in humans?

9) Is the cell-culture isolated NCoV-EMC the 'correct' wild-type viral sequence we should work on?

10) Is the virus adapting to the human population and if so, in what way and how could that impact pathogenicity/transmissibility?

BONUS question:

11) What are we going to about it apart from sit back and wait? 

Updated 2nd April 2013 from Martin Enserink, Matt Frieman and Helen Branswell

12) How similar is EMC to SARS during infection of the human airways?

13) What proteins/genes encoded by EMC inhibit - however effectively - the human innate immune response?

14) Why doesn't EMC replicate in lab mouse strains? (Apparently it doesn't)

15) What epidemiological studies are being done?

16) Is there an intermediate 'amplifying' host?

17) When did EMC first infect humans?

18) How do humans get infected?

and the clincher:

19) Why don't we know the answers to the above questions already?

Three thoughts on the novel coronavirus cell line study

Sometimes a paper is published and the real-world applicability of the study isn't easily concluded or communicated from the results. Yet despite that, these inferences spread among the media and can result in feelings of confusion, panic and dread when the public are faced with the prospect of a virus more pathogenic than the SARS coronavirus was.

This happened recently following the publication of a paper in the Journal of Infectious Diseases (Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications on disease pathogenesis and clinical manifestation) It's OA too so go have a look. There's also a very good accompanying editorial outlining the issues with drawing clinical conclusions from these data. 

A number of news storys and tweets were communicated concluding that this virus is 'more deadly' than hCoV-SARS, which could only replicate in a few cell lines or does the study even provide evidence that the virus can replicate in many different tissue types? There was however a more muted story in CIDRAP. Can they really say that from their data?

Basically the Hong Kong group used the isolated novel coronavirus (hCoV-EMC) from Ron Fouchier's lab and infected a wide range of cell lines with one infectious virus particle per cell and measured production of viral RNA (I think the genomic positive sense strand) on day 0, 1 and 3 following infection as well as nucleoprotein protein expression as markers of replication and concluding from this viral tropism in a human person. From this they showed that the virus could replicate in nearly every cell line tested and could replicate their genome up to five logs (quite a lot).

MY THREE THOUGHTS:

1) The main issue with this paper is this: these cell lines, although originally human, are all immortalized cancer cell lines characterised by markedly different biological properties when compared to normal human cells of the same tissue/cell type. They can't be readily used a surrogates for normal human tissue/cell types. None were primary cells nor were any even from recently acquired tissue samples from biopsies etc. People have infected primary human airway epithelial cultures with hCoV-EMC - so this can be done successfully - , although it would be more difficult for other tissue types as these cultures haven't been developed. Some of these cell lines used may by chance lack key viral repressors of infection present in normal primary cells, which could skew results from cell culture infection experiments. Plus, a human tissue is not just a single cell type - they are composed of diverse kinds of cells that could together behave much, much differently than cell lines in culture. 

2) The pathogenesis and spread of virus relies on the complex interaction with the human immune system in a tissue specific manner. For example, the hCoV-EMC virus may never escape the human respiratory tract because tissue-resident immune cells and the innate immune system cripple virus replication before it can spread systemically in blood or lymph.

3) Virus spread and tropism also relies on physical cell-cell interactions. For example, measles and other paramyxoviruses gain access to diverse tissues in the human body including the brain and kidneys via infection of immune cells residing in near-by draining lymph nodes or those present in sites of primary replication like the lungs. If hCoV-EMC can't do this nor survive and persist in the blood stream/lymph then how is it go systemic?

All these processes can and should be modelled in some way in the lab but certainly not only through these basic cell culture infection experiments. And I should add that this study doesn't prevent others from doing so and encourage other groups across the world to look into this. The complex interactions of emerging viruses with all cell/tissues/biological processes should be investigated! However, that will require further work in more refined models or animal studies. 

One investigation that would prove extremely useful and answer these questions would be the pathological assessment of banked autopsy material from the fatal cases in the UK (this had been done in SARS). Assessment of the distribution of viral antigen could be used to infer virus tissue/cell tropism and point us in the direction of where and what the block or inhibitory factors act to limit virus transmission/severe pathogenesis like that seen with SARS. 

N.B - the idea for this post came from below.

Over twitter I took part in this brief discussion begun by Laurie Garret's tweeting of the link to the study:

I was probably over critical saying it was 'horribly flawed' - the study and science was OK (though see Matt Frieman's - who is a coronavirus group leader in the U.S - comments below) but it is the clinical conclusions that can be drawn that would be flawed if we took this as evidence that hCoV-EMC is more pathogenic than the SARS virus (it clearly isn't).

Then it was pointed out that there was an informative editorial accompanying the article:

HIV finds a cellular door knob - the SIGLEC1 story

This provides some background/analysis of the upcoming #microtwjc week 18 paper on HIV/SIGLEC-1 interactions

Viruses are classed as 'obligate intracellular parasites' and so they have to get inside a host cell, whether they are bacterial, archaeal or eukaryotic. In the case of mammalian viruses, which I have the most experience in, this is a key aspect of how viruses infect, cause disease and transmit themselves from one host to another in a population. In fact it is probably the most important event in the virus life cycle (here's a great link describing the replication cycle of HIV).

Example: Viruses like influenza that really only get inside lung cells will be respiratory transmitted and may cause lung diseases while viruses like HIV that get inside your immune cells and find themselves rushing around your blood stream will only spread via contact with bodily fluids. This is the same for every other human virus in existence.

However one major obstacle to getting inside your cells is the cell membrane, which is impermeable to particles the size of viruses. The virus must coax or force its way into the cell cytoplasm where it can begin its replication cycle and make new virus particles.

This is what a virus has to contend with: the plasma membrane. But what molecule on the surface will it interact with?

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

Buller et al. 2012

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*

This 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

It 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.