Vaccines and viral evolution

This is the first time in ten years that I haven't had an exam around summer-time. It feels odd, everyone around me is either exam-stressed or post-exam-relaxed, it's turning to summer and there's a definite final term feeling but this time I'm not really part of it. It's been an interesting year this year, since January I've not been involved in any part of research science, other than writing about it.

However luckily I'm still surrounded by lectures, seminars, talks and various other interesting stuff which no one seems to mind me occasionally turning up too. Seeing as I haven't written much about virus's lately I headed over to a talk the other day about Marek's disease, which is caused by a Herpes Virus and has a rather devastating affect on chickens.

It makes them stand like this :(

It started back in the sixties, when rather a lot of chickens suddenly started dropping dead, sometimes up to 50% of all the stock in a large barn. Bear in mind these weren't the happy pecking-around-shrubs-of-grass chickens that feature on the front of free-range eggs, but rather a lot of chickens quite closely packed inside a big barn. The cause was found to be MDV - Marek's disease virus. After a lot of work a vaccination was found and given to all the chickens. Over $2 billion was saved by this, and the chickens were able to survive, right up until they got slaughtered for food.

But then, around the 1980s the disease suddenly reared it's head again, this time in a far more virulent form imaginatively labelled vvMDV (which stands for very virulent MDV). More research, another vaccine, and the deaths stopped.

Until just before 2000 when the virus evolved again into an even more virulent strain called vv+MDC, which means exactly what you think it does. Another vaccine was made (called Rispens) but at this point it was becoming fairly clear that this virus was behaving oddly. Three times it had changed, becoming more and more deadly each time:

Image from the presentation slides showing the development of new strains with an increase in virulence.

This is not normal behaviour. Virus's rely on their hosts, they can't replicate, survive or do anything without a host cell, which means they have a vested interest in keeping the host alive. If anything, viral strains should evolve to become less virulent; a virus that kills the host will be a virus without a host and is therefore less likely to survive and propagate than a virus with a host.

It turns out in this case that the evolution of increase virulence is down exclusively to the way the vaccine interacts with the virus. The virus works by being inhaled into the chickens lungs, getting into the cells of the immune system (B and T cells) and causing a latent infection of the lymphocytes (T cells). Virus cells can also work their way to the epithelial cell of the feather follicles and will shed from under the feathers, thus keeping the virus in circulation.

In an ideal situation a vaccine would produce what is known as "sterilising immunity", where use of the vaccine kills all viruses dead. This is how almost all human vaccines work. With the Marek's disease vaccine however, the virus was not killed completely, but could still replicate and shed from the feathers. This means that the virus was still within the system, able to change and evolve. The vaccine however, does make the virus less likely to spread, which means that a more virulent form that the vaccine does not protect against is able to outcompete the less virulent strain. Because the chickens are all in very close proximity, and because there are a lot of them, the more virulent strain can spread much faster throughout the population. With normal chickens, in small isolated populations this would not happen as a virus that virulent would run out of host and die out.

This creates a paradox - vaccines are needed to stop the chickens getting the virus, but at the same time use of the vaccine is creating an evolutionary environment in which a more virulent virus can grow. There are some responses to avoid this though. Firstly, to develop a virus that produces sterilising immunity, i.e that kills all thee virus dead. Secondly, to allow the chickens more room and freedom to stop the virulent strains spreading so quickly. And while this second solution sounds like every animal-rights campaigner's dream, remember that it only really works if very few people in the world eat chicken. In reality there are lots of people, and a limited space for chickens - eating chickens which have lead a happy healthy life is a privilege for a few, not the reality for the majority.

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Witter RL (2001). Protective efficacy of Marek's disease vaccines. Current topics in microbiology and immunology, 255, 57-90 PMID: 11217428

Witter RL (1997). Increased virulence of Marek's disease virus field isolates. Avian diseases, 41 (1), 149-63 PMID: 9087332
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Plant Defence 3 - Acquired Resistance

In posts one and two of this mini-series I explored how plants can defend against bacteria by releasing dangerous chemicals and by killing off cells. This post looks at how surviving one bacterial attack can make plants more able to survive subsequent ones with both local and systemic acquired resistance.

Locally acquired resistance is the simplest to manage, and provides a clear advantage. If cells have been attacked once it makes sense to defend them in case of a second attack. Plants achieve this by strengthening the cell walls in cells that have survived the bacterial attack. Experiments adding elicitors (bits of bacteria that stimulate the plant pathogen receptors) to plant cells showed that proteins in the cell wall became oxidatively cross-linked as they sense the bacteria. Interestingly the molecule responsible for this is hydrogen peroxide, one of the molecules also involved in the cell-death response discussed in post two. If it doesn't kill the plant, it makes it stronger.

The cell membrane is the blue box at the bottom, whereas the cell wall is the light blue rods in the middle. It is the cell wall which is strengthened. Image from wikimedia commons.

This response is all very well for plant cells which happened to be near the site of infection, but what about the rest of the plant? Is it possible for cells on the other side of the plant to be warned and ready for a pathogen attack? Despite the inability of plant cells to move, the answer surprisingly is yes. Cells at the site of infection can release a chemical called salicylic acid which moves through the plants vascular system (the system which also delivers sugars and other important nutrients to all parts of the plant).

The chemical structure of salicylic acid, which is chemically similar to the active component of aspirin.

Following an infection, the levels of salicylic acid were found to rise dramatically in cells around the zone of infection, before spreading through the rest of the plant. This isn't a species specific response either but one found in many different species; grafting parts of one plant onto another did not stop either plant from acquiring resistance. In response to the salicylic acid signal cells start accumulating small amounts of hydrogen peroxide, which can lead to the same cell wall strengthening seen around the area of infection.

As well as salicylic acid it has also been suggested that infected areas of the plant can release the volatile molecule methyl salicylate, commercially known as oil of wintergreen. Rather than travelling through the plant this signal is airborne, allowing transmission not just to other parts of the plant, but to neighbouring (and therefore likely to be related) plants as well. As the only difference between these two signalling molecules is the addition of a small CH3 group, the methyl salicylate can easily be converted back into salicylic acid once it reaches the cells where it can cause the same downstream response.

If anyone was wondering quite why I've suddenly been into plants part of the reason is that the BBC is showing a program called "Botany - a blooming history" and I've been catching the episodes. Despite the slight naffness of the title, it's actually a really good program showcasing experiments, personalities, and the scientific method as it unfolds the history of plant science. You can catch the episodes here on iPlayer.

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Brisson, L., Tenhaken, R., & Lamb, C. (1994). Function of Oxidative Cross-Linking of Cell Wall Structural Proteins in Plant Disease Resistance The Plant Cell, 6 (12) DOI: 10.2307/3869902

Durrant, W., & Dong, X. (2004). SYSTEMIC ACQUIRED RESISTANCE Annual Review of Phytopathology, 42 (1), 185-209 DOI: 10.1146/annurev.phyto.42.040803.140421

Shulaev, V., Silverman, P., & Raskin, I. (1997). Airborne signalling by methyl salicylate in plant pathogen resistance Nature, 385 (6618), 718-721 DOI: 10.1038/385718a0

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Plant defence 2 - Honourable Suicide

The first post of this mini-series covered how plants can defend themselves against bacterial attack by releasing chemicals, either on a regular basis or as a specific response to the attack. This post will explore the hypersensitive response, which allows plants to rapidly kill of cells around the area of infection, starving the bacteria of nutrients to prevent it spreading. The end result is a small area of dead plant matter, with the rest of the organism unaffected.

One of the main differences between plants and animals that I flagged up in the last post is that plant cells don't move. The use of the hypersensitive response shows another; plants have a very non-determinant structure. Animals will grow towards a clear well defined shape and once they get it, they stick with it. Your body does change as you grow older, but it's not about to grow an extra leg. Plants on the other hand may have determinant structures within them, such as leaves or flowers, but the overall organism can just keep growing for as long as it needs to. If a leaf is lost through disease, the plant can just grow a new one, or several new ones.

These leaves are expendable. Your arm is not. Image from wikimedia commons.

Because of its non-determinant nature it is a lot easier for the plant to kill parts of itself off in order to stop an infection spreading. One way that the hypersensitive response does this is by the production of large numbers of reactive oxygen species in cells surrounding the site of infection. These include hydrogen peroxide, and various hydroxide and oxygen containing free radicals. Free radicals are species with one unpaired electron and therefore are extremely reactive and extremely dangerous. These free radicals lead to chain reactions that can break down lipids in the membrane, inactivate enzymes and generally roll around like a loose canon causing havoc within the cell.

As well as reactive oxygen species, the cell also experiences large ion fluxes, as potassium and hydroxide ions flood into the cell and hydrogen and calcium ions flood out. These result in the cell releasing any stored toxic compounds it might have (which may also help to kill the bacteria) and may serve to integrate the mitochondria into the process of cell death (see reference one). As mitochondria are crucial in coordinating the programmed cell death of animal cells it would be surprising if they did not play some part in the controlled destruction of plant cells. The actual sequence of destruction varies from plant to plant, but the overall result is the same, and area of dead plant tissue within the still healthy surviving plant.

A leaf infected with tobacco mosaic virus, showing lighter areas of dead leaf interspersed with the green areas of normal growth. Image from wikimedia commons.

The plant hypersensitivity response can (if you want it to) be considered analogous to the human innate immune response, in that it occurs directly in response to a bacterial attack, and it occurs only at the site of bacterial infection. Plants, however, also have ways of making more long-term changes to protect against bacterial attacks in the future both at the site of the old reaction and throughout the whole plant. How the plant achieves this, without any cellular movement, will be the topic of the final post in this mini-series.

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Lam, E., Kato, N., & Lawton, M. (2001). Programmed cell death, mitochondria and the plant hypersensitive response Nature, 411 (6839), 848-853 DOI: 10.1038/35081184

Pontier, D., Balagué, C., & Roby, D. (1998). The hypersensitive response. A programmed cell death associated with plant resistance Comptes Rendus de l'Académie des Sciences - Series III - Sciences de la Vie, 321 (9), 721-734 DOI: 10.1016/S0764-4469(98)80013-9

Taiz, Zeiger, Plant Physiology, third edition Sinauer Associates 2002.
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Plant defence 1 - Deadly Chemicals

Although I've never officially studied immunology, my second year course in Pathology left me with a pretty solid idea of how humans defend themselves against bacterial attack. Even without a university course I've always been vaguely aware of the presence of immune cells; the B and T cells that make up the adaptive immune system, the clotting response, and the symptoms of inflammation around the site of infection.

How plants responded to bacterial attack was still a complete mystery though. One of the main things that distinguishes plants from animals is that animal cells are a lot more motile, they can move through the body. Animal cell movement is crucial during the development of the embryo and even once the body is fully formed cells still rush around the blood stream and slide around in the epithelial layers. The correct functioning of the immune system relies on cells being able to do this, dendritic cells and macrophages will pick up bits of bacteria at the site of infection and go running back with them to the lymph nodes which will start organising the best way to deal with the infection.

A macrophage in the lungs, from Wikimedia commons. The macrophage engulfs bacteria and eats them, which requires it to be able to move.

With a few odd exceptions plant cells do not move. Not at all. There is no movement of cells during the seed development, and even the movement of plants towards sources of light and water is caused by cells growing rather than moving. How then does the plant respond and react to bacterial infections?

There are several different ways, which is why this is a three-part post series:

1-Deadly Chemicals

2-Honourable Suicide

3-Acquired Resistance.

Part One: Deadly Chemicals

One of the simpler ways to remove a bacterial infection is to release a chemical that is harmful to the bacteria. There are quite a lot of plants that produce antibacterial products as normal secondary metabolites, an example of which is saponins, a group of compounds which have soap-like properties. As saponins are lipid soluble they can break up bacterial membranes by binding to sterol compounds within the membrane and disrupting the structure. Studies done on oats (reference one) have shown that reducing the natural levels of saponin made the oat plants much more vulnerable to fungal infections.

Rather more excitingly, plants can also release certain chemicals in response to a bacterial attack. When bacteria attack plants have been shown to release an assortment of hydrolytic enzymes - glucanases, chitinases, etc that break down cell walls and membranes. These are known as pathogenesis-related proteins as they are specific to bacterial or fungal attack. One of the better researched is a group of chemicals called phytoalexins. In normal conditions neither the phytoalexins themselves, nor the enzymes used to make them, are found within plant cells. It is only after a microbial invasion that the enzymes are transcribed and translated and the phytoalexins synthesised.

In order to respond specifically to bacterial attack, the plant needs to be able to recognise bacteria as invading elements. Like many animals, plants have what are known as "Toll-like receptors" that recognise bacterial pathogen molecules (which in animals are referred to as PAMPS Pathogen-Associated Molecular Patterns but in plants seem to be called elictors) such as bits of protein and polysaccharide fragments from the bacterial cell wall. [EDIT - I have since been informed that PAMP is used quite widely among plantscis now as well]

Comparison of the plant and animal TOLL receptors. The blue and red lines are the receptors, and the blobs attached to them are the bits of pathogen. The yellow boxes labelled PK stand for 'protein kinase cascade' which carries the message through the cell to turn on the genes required. Diagram adapted from reference two.

By recognising pathogens as they invade, the plant cells can launch a deadly chemical attack against them, without requiring any movement. None of this requires the cells to travel around, and until the bacteria develop resistance to the chemicals being used, it can be highly affective. Chemical warfare however, is only one of the strategies that plant cells can adopt to protect themselves against invading microorganisms, and my next post will cover the second - depriving the bacteria of valuable nutrients by committing cellular suicide.

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1) Papadopoulou, K. (1999). Compromised disease resistance in saponin-deficient plants Proceedings of the National Academy of Sciences, 96 (22), 12923-12928 DOI: 10.1073/pnas.96.22.12923

2) Nürnberger, T., & Scheel, D. (2001). Signal transmission in the plant immune response Trends in Plant Science, 6 (8), 372-379 DOI: 10.1016/S1360-1385(01)02019-2

3) Taiz, Zeiger, Plant Physiology, third edition Sinauer Associates 2002.

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E. coli the enemy

It's been in the news a lot, but for those who've missed it there's a particularly deadly strain of E. coli that has reared its head in Germany. The source of the bacteria is not yet known although both cucumbers and beansprouts have been blamed, and German authorities are advising people to stay away from raw leafy vegetables. Vegetables don't usually harbour their own strains of E. coli, but the thought is that animal manure (which most definitely does contain E. coli) used to fertilise the vegetables may be carrying the deadly strain.

This plate is more deadly than you can possibly imagine

I talk about E. coli a lot in my blog as I use it for most of my experiments. The strain I use is called K12 and is completely harmless to humans. It's also used in sterile conditions and not allowed to leave the lab. The strain that's causing panic in Germany is called O104:H4 which is an enterohemorrhagic strain which can cause bloody diarrhoea and also attack the kidneys. Working from news sources (as there don't seem to be any papers out yet!) it seems to have picked up the DNA with a kidney damaging toxin as well as having proteins that help it to stick strongly to the intestinal cell wall. Other bloggers have looked at this in more exact detail (and shared their findings, which is pretty awesome), including which parts of the DNA have changed in this new strain.

The labelling isn't just arbitrary either, it tells you exactly what antigens the bacteria is carrying, and antigens are the things that the immune system recognises to help your body attack the disease. There are three different types of antigens in the E. coli species; O (in the new deadly strain), H and K (in my lab strain). The letter and number after the : mark "O104:H4" refers to the flagella antigen. Strains which share the same antigen types will have similar patterns of virulence.

As well as trying to understand the genetic code and how this strain differs from other deadly and non-deadly E. coli, scientists are also trying to find exactly where the bacteria are coming from, testing vegetable sources in an attempt to isolate it. Both cucumbers and beansprouts have now been tested for the strain isolated from patients, and both have shown negative results.

Understanding where the deadly strain arises from is less useful in terms of the scientific investigation, but crucial for stopping the disease from spreading, and helping to understand how to avoid such strains developing in the future.

If you're worried about the vegetables you're eating then the best advice I can give is to cook before eating. E. coli are not able to withstand the high temperatures of cooking and unlike bacteria such as clostridium they do not release toxins onto the food. Clostidium produce exotoxins directly onto your food whereas E. coli produce endotoxins once they get into the body. For the more sciencey minded, it's Gram positive bacteria which tend to produce exotoxins, whereas Gram negatives produce the endotoxins.

E. coli image from Wikimedia commons

It's only once the E. coli enter your body that they start producing 'toxins' and these toxins are usually parts of the bacteria as they are broken down by the immune system. If you manage to destroy the bacteria before eating the food, then it will be pretty much safe to consume. Note that this technique does not work for exotoxins, which are actively secreted by the bacteria (Gram positives like secreting things) onto food before you eat it.

Studying bacteria does sometimes feel like a little niche set slightly apart from the real world, which is full of lumbering eukaryotes making complex non-rational interactions. News like this an interesting reminded that bacteria don't just affect health, but also have strong economic and political implications. Spanish cucumber salesmen are trying to sue Germany, Russia's getting all smug about EU health regulations, and German tourism is being affected. All because of E. coli.

Molbio Carnival #11

This is the eleventh edition of the Carnival of Molecular Biology, a travelling goodie-bag dealing with all things small and cellular. When this carnival was first started up I must admit I had more of a hope than a certainty that enough people would be interested in the world of the small and cellular for it to continue, but we've passed the tenth edition, and it's still here!

Long may it remain

For this edition, we'll start off with the all important question, what actually is a biochemist? And is it significantly different to a chemical biologist? My reasoning on this is clear; a biochemist is a biologist who likes the chemical side of things, and a chemical biologist is a chemist who is fascinated with biology. They both may work in the same lab, but will have different training backgrounds and different ways of seeing the world. Chris Dieni gives a more thorough explanation of this over at BenchFly.

Science works by experiment, which is why I'm happy that we have two posts this carnival covering experimental techniques used to explore the intracellular landscape. The Biotechnology blog takes us through the technique of 3-dimensional cellular arrays which build up a picture of the entire cell, rather than just using thin slices through the cell. Psi Wavefunction goes deeper and looks at how to study the DNA within cells, and as she works with protists, it's some very strange DNA indeed.

Lineup of DNA, from Psi's post

As well as biologists and chemists, engineers tend to get involved in this molecular-biology gig and when they do they almost inevitably start talking about lego. Lucas from Thoughtomics shows us how life can be thought of as lego blocks, and how planets with life on them could potentially be found, even if the life is not as we know it.

Back in the realm of pure biochemistry is a brilliant post from It Takes 30 which explores how one pathway within the cell can lead to many different outcomes. It focusses on the pathways of p53, an unassuming little molecule that is one of the most important within the cell, as it responds to cellular DNA damage.

From the It Takes 30 post - a diagram many biochemists

are familiar with!

Finally, we'll finish with a last post from Psi, which is part of a multi-post essay about constructive neutral evolution and is well worth reading. As someone who only dashes of quick little posts, it's great to read a well researched longer post, which goes into a good (and accessible!) coverage of an important topic.

That's all for this edition! If you want to get involved (and I strongly recommend it, carnivals are interesting, great publicity, and also quite fun) submit any molecular-biology related posts here. The next edition will be at PHASED and the more posts it has, the bigger and better the carnival will be.

Lab Rat vs Fake Science

For those of you who might have missed my writing, I've written a piece for the Varsity magazine "Not-Sci" section, which examines false scientific claims. My piece is looking at a little Gadget called Totem Confidence, which claims through the power of a CD and a keyring to increase confidence, reduce stress, and reduce your bank balance by £14.97. You can read it here. LINK IS NOW UP AND RUNNING AGAIN.

For those of you who want to promote your own work, or have a desire to read all sorts of other pieces, Lab Rat will be hosting the MolBio Carnival on the 6th. If you've written (or read!) any blog posts that deal with the inner life of the cell then please do submit them it would be great to have loads of interesting posts.

And after that ... the science posting will return with a vengeance, because I have a PhD starting up in a few months, and I want to be a microbiological expert before I head in!