Mystery Rays from Outer Space

Meddling with things mankind is not meant to understand. Also, pictures of my kids

October 26th, 2008

Where did avian flu come from, and where is it going?

Wild geese and rushes - Huang Chu Tsai
Wild Geese and Rushes
(Huang Chu Tsai - 
Sung Dynasty)

Where did avian influenza come from?

The H5N1 avian influenza virus infects mainly birds, but there have been plenty of cases of spread into humans, where it is much more virulent than the ordinary, generic human influenza viruses that sweep around the world each year. H5N1 avian influenza is actually a group of related viruses, not just a single virus; and although it’s now mainly a virus of chickens and ducks, it was originally a pathogen of waterfowl.

When viruses jump from one species to another, they’re usually poorly adapted to their new host. Most often, the new host probably doesn’t even notice the infection. Sometimes the virus may be highly pathogenic, and may even rapidly kill the new host: Ebola virus (bat to human), Sin Nombre virus (rodent to human), and SARS virus (palm civet to human), are all examples of this. But in spite of high virulence, the virus isn’t necessarily well adapted to the new host; it doesn’t transmit effectively from one individual to another, and the infection burns itself out, only sustained by repeated jumps from the original population into the new one. That’s the phase we’re in with avian influenza.

SARS virus, though, is also an example of another viral phenomenon. The virus may be poorly adapted at first, but viruses (with their 24-hour generation time and in many cases their high mutation rates) evolve fast. What’s more, though the new host may not be a good fit in some ways, the entire population should be immunologically naïve — that is, there should be no immune resistance to the virus in the new host, in contrast to the original host, which typically would have a fair bit of resistance spread around the population. If the virus can adapt to the new host at all, there’s a smorgasbord of victims for it to infect. SARS virus jumped from palm civets into humans a few times, and then it rattled around in humans for a bit, gradually adapting and accommodating itself to humans instead of civets, until it was — well, not a very good human pathogen, but at least it was adequate at transmitting itself; it was capable of a sustained epidemic with further input from the parental civet virus.

Not much is known about how viruses acclimate to new species. Since it’s generally assumed that the next pandemic influenza outbreak of humans will come from the H5N1 virus once it’s become better adapted to humans, it’s of obvious interest to learn how it made its earlier jump to become adapted to chickens and ducks. A recent paper in PLoS Pathogens1 tracks the virus back to its roots.

Rooster (Historiae animalium lib. I.)Influenza viruses have a segmented genome — that is, the RNA that makes up the viral genome is split into eight separate pieces. If two influenza viruses infect the same cell, then when the new pieces of RNA get packaged up to make a virus, you can get reassortment — the progeny viruses can contain RNA segments from two different parental viruses. Did H5N1 adapt to domestic birds through reassortment with a well-adapted chicken virus, or did it make the jump all at once, as a single virus, and later on become better adapted?

Vijaykrishna et al looked at a lot of virus sequences and concluded that the virus did originally jump to ducks from migratory waterfowl as a single already-formed virus. The common ancestor of all the present H5N1 viruses probably formed, in migratory waterfowl, around 1994 — a couple of years before H5N1 was detected as a highly virulent pathogen of domestic ducks, which was in 1996.

After it was introduced into ducks, then new reassortants did arise — probably as the virus began to acclimate to its new hosts:

Analysis of virus population dynamics revealed a rapid increase in the genetic diversity of Gs/GD lineage in poultry in China from mid-1999 to early 2000. This corresponds with the period when each of the major Gs/GD-like H5N1 variants or sublineages diverged and subsequently became widespread in poultry throughout China. It is likely that combined strong ecological and evolutionary factors led to this rapid increase in diversity, namely, the spread of the virus through large, immunologically naive poultry populations…1

The authors suggest that China is a particularly good greenhouse for new H5N1 viruses to arise, probably because of the large and frequently connected population of domestic birds:

… transmission of these reassortant viruses within large highly connected populations of duck and other poultry species results in frequent interspecies transmission and genetic drift. Therefore, it is likely that this process selects for relatively fit viruses with a broad host range which are subsequently exported to other geographical regions. It is interesting to note that further reassortment has not been observed once those H5N1 viruses were transmitted out of China. We suggest that host population structures elsewhere may not result in the same intense multi-species transmission we observe in southern China.1

The selection for “relatively fit viruses with a broad host range” is the main concern here, I think. The next pandemic is likely to come out of China.

  1. Dhanasekaran Vijaykrishna, Justin Bahl, Steven Riley, Lian Duan, Jin Xia Zhang, Honglin Chen, J. S. Malik Peiris, Gavin J. D. Smith, Yi Guan, Ron A. M. Fouchier (2008). Evolutionary Dynamics and Emergence of Panzootic H5N1 Influenza Viruses PLoS Pathogens, 4 (9) DOI: 10.1371/journal.ppat.1000161[][][]
October 19th, 2008

Immunodominance and latency

MHV68 (Stevenson)
MHV–68 exiting an infected cell (Stevenson lab)

Some viruses burn through their hosts like a flamethrower, blasting past defenses for a brief shining moment and then hurtling on through the next victim. Other viruses slip in, delicately negotiate an understanding with the immune system, and set up a long-term relationship with the host. Herpesviruses do the latter, and to me, at least, they’re more interesting than their less restrained cousins.

Herpesviruses set up long-term, usually lifelong, latent infections in their hosts. The cells that harbor the latent infections depend on the precise virus; gamma-herpesviruses like Epstein-Barr virus (EBV) typically go latent in lymphocytes. EBV only infects humans, so it’s kind of hard to do actual experiments (as opposed to observational studies) but a possible model for EBV is a mouse gamma-herpesvirus, mouse herpesvirus 68 (MHV68). I’ve talked about this virus before here, in the context of immune evasion; MHV68 has a gene that apparently allows it to avoid T cell recognition in the initial stages of infection, and this immune evasion in the early stages of infection helps determine the extent, and stability, of the latent phase. In the latent phase, though, the immune evasion molecule might not be all that helpful. What regulates the numbers of persistently-infected lymphocytes?

Immunodominance is another theme I’ve talked about several times here. Immunodominance is the phenomenon in which T cells focus their response on a small number of potential targets. A typical virus — especially a large virus, like a herpesvirus — might have hundreds or thousands of potential T cell targets (“epitopes”), according to the simpler prediction programs; but in practice, only a tiny handful of those hypothetical epitopes are actually recognized efficiently by T cells in the host. The molecular mechanisms that determine immunodominance aren’t well understood, and in many cases the importance of immunodominance isn’t clear, either. In the case of HIV, and perhaps a couple of other viruses, there’s evidence that immunodominance affects the course of infection, but there aren’t many such clear examples.

MHV68 is apparently another example. A paper from Marques et al.1 shows that in one particular mouse strain, T cell recognition of a single epitope of MHV68 is critical in determining how much latent virus hangs about in the infected mouse. Mutating that single epitope (so that it was no longer recognized by T cells) cranked up the level of latent infection dramatically.

MHV68-infected lung
Lung section from a MHV68–infected mouse

In this case, the immunodominance is not so mysterious; there aren’t all that many genes expressed during latency (pretty much by definition) and so there aren’t many possible sources for the dominant epitope. Still, it’s surprising (to me) that a single epitope can have such a dramatic effect on the pathogenesis of the virus; I can’t think of very many instances of that. An obvious question is whether this is unique to this particular mouse strain (because mouse strains typically see different epitopes) or whether this is a general phenomenon. That’s a really hard question to answer, but the authors point at a little bit of circumstantial evidence: Apparently the gene that’s the source of the epitope shows evidence that it’s under more selection than are the neighboring genes.

So the authors’ model for this virus’s pathogenesis, as I understand it, would be something like this: In the initial infection, the virus expresses many genes, and probably has many targets for the immune system. But some of the genes it expresses are immune evasion genes, which dampen but don’t eliminate the immune response, allowing the virus to get access to the lymphocytes it needs for latent infection. The virus constantly, though slowly, reactivates, so that there should be fewer latently-infected lymphocytes over time; that’s counteracted by the virus forcing the lymphocytes to replicate themselves at about the same rate. At the same time, though, latently-infected lymphocytes will be also gradually depleted by the immune system. This immune-mediated depletion depends on the virus having a target for the T cells to see; because there are only a small number of latently-expressed genes, there are only a few possible targets for the T cells. Immunodominance (probably) narrows this even more, in this case to a single T cell epitope. Get rid of that epitope and there’s no target at all, so that instead of merely keeping pace with the depletion, the virus can at least temporarily get ahead of the depletion, increasing the latent set-point of the virus.

In the case of EBV, at least, it’s likely that the latent set-point is important in disease, so (if MHV68 really is a model for EBV infection, which is a little controversial) this may be an example of immunodominance determining disease.

  1. Sofia Marques, Marta Alenquer, Philip G. Stevenson, J. Pedro Simas, Ann B. Hill (2008). A Single CD8+ T Cell Epitope Sets the Long-Term Latent Load of a Murid Herpesvirus PLoS Pathogens, 4 (10) DOI: 10.1371/journal.ppat.1000177[]
October 13th, 2008

Immune evasion as a vaccine target

Temetomo repelling smallpox demon
Temetomo repelling the demon of smallpox
(Utagawa Yoshikazu, ca. 1847 – 1852)

I talk a lot on this blog about viral immune evasion. I’m most interested in the ways by which, and the evolutionary reasons for, viral evasion of T cell recognition; but there are lots of other branches of the immune response, and viruses have ways of evading most of those branches. I’m even prepared to say that, all in all, evasion of T cell immunity is probably relatively a minor component of pathogenesis for most viruses.

For one thing, most viruses don’t even bother to evade T cell recognition, as far as we know. Although a handful of viruses, like some of the human adenoviruses, apparently use it, only one virus family — the herpesviruses — seem to have evolved T cell immune evasion as a common, highly conserved function. And to the extent that it’s been examined, which isn’t very far, T cell evasion doesn’t make a huge difference to the virulence of the virus, or otherwise have massive effects on the virus’s ability to replicate. That’s not to say there’s nothing out there1 but all in all, it’s not particularly impressive.

On the other hand, as I’ve pointed out before, there are forms of immune evasion that do have a very large effect on the virus’s virulence. The example I mentioned before was influenza virus. Different strains of influenza virus are more or less able to prevent innate immune recognition; swapping efficient immune evasion into a relatively mild influenza virus turns it into a much more severe pathogen.

Testimonial to smallpox vaccination
“Expositions on the Inoculation of the Small Pox and of the Cow Pock”
 By John Coakley Lettsom (1806)

So is this something we can take advantage of? If there’s increased immune recognition of a virus, then perhaps the virus would make a better vaccine; especially if it’s less virulent at the same time. Frank Ennis’s lab2 has recently demonstrated that this might be a useful approach to vaccine development.

In this case, they used vaccinia virus. Vaccinia, of course, is the virus that was used as a vaccine against smallpox virus. With bioterrorism in the spotlight over the past few years, there’s renewed interest in vaccines against smallpox. Although vaccinia worked very well to eliminate smallpox, it’s not particularly safe; the risk of adverse effects is far higher than is tolerated in most vaccines these days. It was worth the risk to eliminate smallpox, but it’s not so clear that the tradeoff is worth it for the purely hypothetical chance of bioterrorism. Accordingly, there’s a lot of interest in developing safer, yet still highly effective, vaccinia-based vaccines. The Ennis lab approached this by knocking out an immune evasion gene in the virus, and has tested their new strain for safety and immunogenicity.

Safer virus, equal immunity

Note that avirulence and immunogenicity, in many cases, have to be balanced against each other. Immune responses tend to be stronger for more dangerous pathogens, because the inflammation associated with cell death and large numbers of viruses stimulates more cytokines, and triggers a more potent immune response. Weakening the virus so that it can’t replicate as well drops the danger, but also drops the stimulation to the immune system. The potential advantage of knocking out immune evasion genes is that any given amount of virus may be more effective at inducing an immune response, because the usual dampening effect of immune evasion is gone.

That’s precisely what Mathew et al found. The knockout virus lacking an immune evasion gene “N1L” is less virulent — it causes less disease, and replicated to much lower levels. This was already known from previous experiments by Geoffrey Smith’s group,3 and, long ago, by Bernie Moss’s lab4 but here they tested the virus in a more biologically meaningful way, by intranasal infection (a better match with the way smallpox naturally infects) and measuring replication in the lungs. The knockout virus only reached 1/10,000 the level of infection as did wild-type vaccinia. (Irritatingly, they don’t specifically compare symptoms, such as weight loss, between the wild type and knockout virus; but at least at one dose the knockout virus didn’t kill mice while the wild-type virus did.)

This was not a particular surprise, but the interesting part was the immune response. Even though there was 10,000 times less virus present, the T cell response to the knockout virus was equivalent to that against the wild-type virus:

Our data indicate that the attenuated vGK5 virus is immunogenic and elicits robust immune responses that are comparable to the wildtype VACV-WR when administered by multiple routes.2

It’s worth repeating that this immune evasion molecule does not specifically affect the T cell response per se. It affects recognition upstream of the T cell response, at a very early point after infection. By reducing this innate immune recognition, the N1L gene reduces overall inflammation. Presumably, the increased inflammation associated with the knockout virus counteracted the reduced numbers of virus, and allowed an equal immune response to a less virulent virus.

A universal solution?

Is this a universal approach that will work with any virus that can evade the innate immune response? It’s certainly worth looking at, but it’s also worth keeping in mind that increasing inflammation is a two-edged sword. Inflammation itself is hazardous, and especially in the lungs inflammation is often the actual killer following viral infection. There’s a well-known study in adenoviruses that concluded that eliminating viral immune evasion increased the virulence of the virus, and this increase in virulence was associated with increased lung inflammation. Although this conclusion is, to my mind, now a very dubious one for reasons I outline here, the principle that inflammation can be dangerous holds. I suspect that using these sorts of immune evasion knockout viruses as vaccine platforms will have to be tested on a case by case basis.

  1. For example: Stevenson PG, May JS, Smith XG, Marques S, Adler H, Koszinowski UH, Simas JP, Efstathiou S (2002) K3-mediated evasion of CD8(+) T cells aids amplification of a latent gamma-herpesvirus. Nat Immunol 3:733–740.[]
  2. Anuja Mathew, Joel O’Bryan, William Marshall, Girish J. Kotwal, Masanori Terajima, Sharone Green, Alan L. Rothman, Francis A. Ennis, Linqi Zhang (2008). Robust Intrapulmonary CD8 T Cell Responses and Protection with an Attenuated N1L Deleted Vaccinia Virus PLoS ONE, 3 (10) DOI: 10.1371/journal.pone.0003323[][]
  3. Bartlett N, Symons JA, Tscharke DC, Smith GL (2002) The vaccinia virus N1L protein is an intracellular homodimer that promotes virulence. J Gen Virol 83: 1965-1976.[]
  4. Kotwal GJ, Hugin AW, Moss B (1989) Mapping and insertional mutagenesis of a vaccinia virus gene encoding a 13,800-Da secreted protein. Virology 171: 579-587.[]
October 8th, 2008

Nobels, part II: Chemistry ’08

Jellyfish (by webmink)
“Jellyfish”, by webmink

GFP? Really?  

The Prize for PCR, back in the 1990s, helped establish that the Prize can go to technical advances, as well as those that have deeper theoretical implications.  I didn’t have any problem with PCR, seeing as how the technique has enabled a vast range of advances in biology:  It’s safe to say that without PCR, biology in general would look completely different today.  

But GFP? Maybe I’m missing something, but for all the widespread use and utility of GFP and its friends and derivatives, is it really such a fundamental advance that it deserves the Nobel?  And in particular, for Chemistry?  Are there new theories or concepts in chemistry that wouldn’t have arisen without GFP?

October 6th, 2008


The Prize to Montagnier/Barre-Sinoussi surprises me a bit, though I think it’s well-deserved.1  Conventional wisdom back in the 1990s, at least with the people I hung out with, was that Montagnier wasn’t going to get a Nobel. The reasoning was that the Committee would feel the need to share any Prize with Gallo, but that Gallo had eliminated himself with his behaviour. (I’m not making judgements here, just passing on the general feeling at the time.)

Now it seems that the committee is OK with rewarding Montagnier and Barre-Sinoussi without even a mention of Gallo. I think that would have been unthinkable back in the mid-1990s, and shows how much times have changed.

On the other hand, I think the Committee was aware of the question. The reason I think this is that they rewarded three people (the maximum) even though HIV and HPV are at best an awkward marriage. The research leading to each discovery is completely distinct. Without going through the list of past winners, I can’t think of a single Prize going to such disparate lines of work — usually it’s to three people who worked on overlapping or closely-related work. Here the only relationship is “viral disease”, which is a pretty broad catch-all.

Since there’s not going to be much argument over zur Hausen’s merits, that makes it harder to argue for Gallo — they end up having to argue that zur Hausen would have to be removed from the Prize, which isn’t going to happen.

Sneaky people on the Nobel committee.

  1. I posted this as a comment on Sandwalk earlier, but I decided to include it here as well.[]
October 6th, 2008

Sex, stats, and sweat

Sweaty t shirtIt’s been suggested for a long time that mice select mates by smelling MHC types, perhaps in the urine. MHC is by far the most variable region in vertebrate genomes, so this would offer a way for mice to avoid inbreeding: The more related the mice, the more likely they are to be similar at the MHC, so selecting a different MHC will help avoid inbreeding.

Partly as an argument by analogy, and partly through some rather poor-quality experiments, it’s also been argued that humans select mates the same way — that differences in MHC type make a partner more desirable. These are the notorious sweaty T shirt experiments that most people seem to have at least vaguely heard of.

I started off very skeptical about the human claims, because the quality of the experiments has, as I say, tended to be poor. There have been small numbers of people, indifference to alternative explanations, and a lot of post hoc hand-waving. (If the preferences turned out to be reversed, why, it was because the female was near her period, or something like that.) I think that most people who have actually looked at the data have had similar reservations, but that hasn’t stopped the concept from becoming pretty well known.

MHC & mate choice I became even more skeptical about the human experiments as I learned more about the mouse data. The evidence for MHC as a mechanism for avoiding inbreeding turned out to be relatively weak, or at least inconsistent (see here for my first discussion); and recently a paper that I found fairly convincing (discussed here) suggested that MHC is not in fact used by mice in this way at all — rather, a much more plausible, highly variable family of molecules called “major urinary proteins” (MUPs) are the source of the anti-inbreeding odor in mouse urine.

Much of the interest in human MHC and sex has been driven by the mouse observation, so I think that if mice don’t use MHC to select mates, then likely humans don’t, either. Still, it remains possible, even probable, that difference species use different methods to select mates. And since humans don’t even have variable MUPs (as far as I know) MHC remains in the chase.

A recent paper1 tries to look at this in a more objective manner, using genome-wide data on couples. Unfortunately the numbers are still quite small (just 30 couples each from a European-American subset, and an African subset) and the results remain slightly ambiguous. Their conclusion was that

African spouses show no significant pattern of similarity/dissimilarity across the MHC region … We discuss several explanations for these observations, including demographic effects. On the other hand, the sampled European American couples are significantly more MHC-dissimilar than random pairs of individuals … This study thus supports the hypothesis that the MHC influences mate choice in some human populations.

So, heads we win, tails you lose, because even though their hypothesis was invalidated overall, some post-hoc wiggling (“demographic effects”) lets them dismiss the data they don’t like.

I’m still pretty skeptical about any real effect from MHC on mate choice. I’m willing to be convinced otherwise, but it’s going to take a larger and more rigorous study than this one to make me interested.

  1. Raphaëlle Chaix, Chen Cao, Peter Donnelly, Molly Przeworski (2008). Is Mate Choice in Humans MHC-Dependent? PLoS Genetics, 4 (9) DOI: 10.1371/journal.pgen.1000184[]