Field of Science

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.


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


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'.