Mystery Rays from Outer Space

We’ve been promised that as genome sequencing becomes faster and simpler, we’ll start seeing practical dividends as well as parlour tricks like sequencing Watson’s genome. Some of the dividends are already paying out, as a paper in the latest PLoS Pathogens¹ shows.

Probably most of you remember the outbreaks of foot-and-mouth disease in Britain in 2001, and again last year. FMD is a virus that affects many hooved animals; it’s not usually fatal, but causes productivity loss. FMD outbreaks are economically devastating, because aside from the productivity loss many countries, that are free of the disease, will refuse to take meat or other agricultural products from outbreak areas. The goal of FMD management, then, is to keep it away, and if it ever hit, to contain it and slaughter all infected and potentially-infected animals.

The 2001 outbreak in Great Britain came from outside the country. The 2007 outbreak, though, was clearly from a local source: The FMD research lab in the Institute for Animal Health (IAH), Pirbright, Surrey. The latest paper discusses the epidemiology of that outbreak, and how they used whole-genome sequencing to track and predict sites of FMD.

(This is timely, because the US is planning to move the sole American FMD research center, now on Plum Island, to the mainland. There’s obvious concern that the virus could escape from containment within research labs and infect neighboring animals, causing the first American FMD outbreak since 1929. I am not particularly knowledgeable about the field, but I have to think that, at best, the timing of the planned move is unfortunate.)

FMD is caused by a picornavirus, the same broad family as polio and cold viruses. Like those viruses, FMD mutates rapidly, traveling around as a quasispecies cloud. The clouds can be easily divided into 7 broad groups, and within the most common serotype (O) there are 8 distinct subgroups (see the map² to the right [click for a larger version] for their geographical distribution).

The FMD genome is 8134 nucleotides long, and the sequence analysis that has been used for epidemiology like the 7 different topotypes has been based on no more than 8% of that length — the VP1 gene, usually. That’s enough to track high-level changes, because of FMD’s rapid mutation rate:²

the rate of evolution is approximately 1% per year …. If the concept of a constant evolutionary rate is accepted and there are no constraints on virus evolution then it would expected that new topotypes could arise in approximately 15 years. In reality, this extent of evolution probably takes much longer. For example, FMD viruses belonging to the Asia 1 serotype, first identified in samples from Pakistan in 1954 … have not yet exceeded 15% nucleotide difference …

But 8% of the genome is not nearly enough to track changes within a single epidemic, like the one in Surrey last year; it simply isn’t long enough to pick up the handful of variations. It was known in the previous outbreak, in 2001, that the information was there in the genome (”virus recovered from closely housed animals can differ by 1 to 2 nucleotides and is likely to pass through a “bottleneck” on passage between farms”).³ The issue was a practical, technological one — being able to sequence entire virus genomes quickly enough to pass back information to people in the field.

By 2007, the technology was there. The people at the IAH were able to sequence genomes from viruses isolated in the outbreak with a fine enough comb to track changes throughout the spread, and fast enough pass information back to the field within 24-48 hours. Their sequencing confirmed that the virus was in fact a lab escapee, because it was almost identical to a couple of lab strains but was different from circulating viruses. ⁴

The 40-odd viral genomes yielded a fair bit of useful information (see the figure to the left for a summary). For example,

The small number of nucleotide substitutions observed between viruses from source and recipient IP suggests that there has been direct transmission without the involvement of other susceptible species, e.g. sheep or deer.

It’s obviously useful to know if there’s a wild-animal reservoir of disease, but an even more important insight came from this work as well.

the virus from IP3b was nine nucleotides different from the virus from IP1b … This is a high number of changes for a single farm-to-farm transmission … and we predicted that there were likely to be intermediate undetected infected premises between the first outbreaks in August and IP3b. … Serosurveillance of all sheep within 3 km of the September outbreaks revealed another infected premises (IP5), on which it was estimated that disease had been present for at least two, and possibly up to five weeks. As Figure 2B shows, IP5 is a likely link between the August and September outbreaks.

I would be interested in hearing from the people on the ground just how useful this information was — for example, were they impelled to search more for an intermediate source based on this information, or did they already suspect it from other, classical ways? But in any case, it’s clear that genomics is capable of pushing epidemiology a lot further in the future.

1. Cottam, E.M., Wadsworth, J., Shaw, A.E., Rowlands, R.J., Goatley, L., Maan, S., Maan, N.S., Mertens, P.P., Ebert, K., Li, Y., Ryan, E.D., Juleff, N., Ferris, N.P., Wilesmith, J.W., Haydon, D.T., King, D.P., Paton, D.J., Knowles, N.J. (2008). Transmission Pathways of Foot-and-Mouth Disease Virus in the United Kingdom in 2007. PLoS Pathogens, 4(4), e1000050. DOI: 10.1371/journal.ppat.1000050[↩]
2. Samuel, A. R., and Knowles, N. J. (2001). Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes). J Gen Virol 82, 609-621.[↩][↩]
3. Cottam, E. M., Haydon, D. T., Paton, D. J., Gloster, J., Wilesmith, J. W., Ferris, N. P., Hutchings, G. H., and King, D. P. (2006). Molecular epidemiology of the foot-and-mouth disease virus outbreak in the United Kingdom in 2001. J Virol 80, 11274-11282.[↩]
4. As far as I know, it’s not yet known how exactly the virus escaped from the IAH. I’ve read what seems to be informed speculation that it may have come from the drains, as decontamination systems designed to prevent that weren’t properly maintained; but I don’t know if that’s true, an educated guess, or mere rumor and guesswork.[↩]

To the extent that I’m a virologist at all, I’m mostly a DNA virus kind of guy, so I can’t give a lot of deep background about noroviruses. I know what everyone knows — noroviruses are a major cause of gastoinstestinal symptoms, especially where people congregate in groups — cruise ships are notorious sites for norovirus epidemics — but also pretty much anywhere; hundreds of thousands of people are infected weekly in Britain at the moment, for example. The virus is a smallish RNA jobbie (a member of the caliciviruses: single-stranded positive-strand RNA, a bit over 7500 bases long). And it turns out to be extraordinarily interesting in its evolution.

This is from
Lindesmith, L.C., Donaldson, E.F., LoBue, A.D., Cannon, J.L., Zheng, D., Vinje, J., Baric, R.S. (2008). Mechanisms of GII.4 Norovirus Persistence in Human Populations . PLoS Medicine, 5(2), e31. DOI: 10.1371/journal.pmed.0050031
They were able to track the sequences of noroviruses involved in epidemics over the past 20 years, and analyzed them functionally. They found two functional changes over time: First, the viruses shift their targets (so that people who are resistant to infection today, may not be in five years time); and second, the viruses drift antigenically, so they avoid the previous year’s immune response.

Both of these evolutionary directions surprise me, at any rate. First, I’m not used to viruses being able to blithely switch their receptor over time; and second, my impression has been that immunity to noroviruses is so weak and transient that the virus wouldn’t need to worry about last year’s immunity to any significant effect.

The receptor thing is apparently because noroviruses use a family of carbohydrates as their receptor; the carbohydrates are variable among the human population, so that:

Variation in the capsid carbohydrate-binding domain is tolerated because of the large repertoire of similar, yet distinct HBGA carbohydrate receptors available on mucosal surfaces that could interface with the remodeled architecture of the capsid ligand-binding pocket.

As for the transient immunity, it seems that I’m a little out of date, though I have company — the accompanying review article in the same issue of PLoS Medicine says:¹

Acquired immunity is not thought to last until a subsequent norovirus season, though a few individuals may acquire longer-lasting immunity. With these factors combined, one might think that immune selection pressure would be rather transient-only heavy at the end of a season-and that an evolutionarily stable strategy for norovirus might be to wait out the summer low season and attack again when population immunity has waned. This is not what Baric and colleagues have found.

It’s true that early studies on noroviruses did show only transient immunity, but apparently a number of recent studies have shown that long-term immunity is possible. ² Critically, in the years following outbreaks of a new norovirus strain, infection rates dropped, suggesting that at least some herd immunity exists.³ That being the case, it’s not surprising that noroviruses evolve to escape from this pressure:

not only does antigenic drift occur in the capsid region of GII.4 norovirus strains over time, but that the variation greatly influences the ability of preexisting herd immunity to neutralize extant strains, based on carbohydrate blockade assays.

Finally, just to make Larry Moran happy, the authors point out that most of the changes in noroviruses over time are due to random drift:

In our analyses, the shell domain appears to be evolving by random drift, as only 5% of changes are informative (i.e., became fixed in the population).

1. Lopman B, Zambon M, Brown DW (2008) The Evolution of Norovirus, the “Gastric Flu”. PLoS Med 5(2): e42 doi:10.1371/journal.pmed.0050042[↩]
2. Lindesmith L, Moe C, Lependu J, Frelinger JA, Treanor J, et al. (2005) Cellular and humoral immunity following Snow Mountain virus challenge. J Virol 79: 2900-2909.
   Siebenga JJ, Vennema H, Duizer E, Koopmans MP (2007) Gastroenteritis caused by norovirus GGII.4, The Netherlands, 1994-2005. Emerg Infect Dis 13: 144-146.
   Lindesmith L, Moe C, Marionneau S, Ruvoen N, Jiang X, et al. (2003) Human susceptibility and resistance to Norwalk virus infection. Nat Med 9: 548-553.[↩]
3. Siebenga JJ, Vennema H, Renckens B, de Bruin E, van der Veer B, et al. (2007) Epochal evolution of GGII.4 norovirus capsid proteins from 1995 to 2006. J Virol 81: 9932-9941[↩]

“Typically, viruses that rapidly kill their host have a very short history, as they rapidly run out of places to reproduce.”

I’m quoting John Timmer from Ars Technica’s Nobel Intent, from a couple of weeks ago. I feel kind of bad about this because I’m only quoting to disagree with him, and I always like Nobel Intent and find it interesting — but this is my most recent sighting of what I think is a very widespread misunderstanding. I commented on it briefly in the thread there (“This is one of those widely-believed rules that’s not nearly as universal as people think. … “), but here’s a chance to expand a bit. ¹

The concept is intuitively satisfying: A pathogen that rapidly and inevitably kills its hosts runs out of new hosts; if the host remains alive longer, then there’s a continuing supply of new hosts; therefore rapidly-lethal pathogens hastily evolve toward reduced virulence. It seems to make all kinds of sense, and there are some famous examples that fit this theory beautifully. Myxomavirus is the type specimen. It also fits with the observation that some of the most virulent virus infections we see are recent introductions into humans — HIV, obviously; also SARS, Ebola, and so on — that may not yet have had time to evolve toward avirulence.Unfortunately, there are also lots of counterexamples to the theory, starting with rabies (a beautifully-adapted virus that is invariably lethal), and aside from myxomavirus there aren’t all that many good examples pro. The reality is probably that evolution toward reduced virulence is a special case rather than a general rule. What’s more, the lay understanding of the theory — viral evolution toward avirulence — has little if any support, and may not occur at all.Everyone² knows about myxomavirus in Australia. Myxomavirus was introduced there in the early 1950s as a biological control agent for the rabbit plague. At first, the virus killed virtually every rabbit it infected (99.8% lethality), reducing the rabbit population by 85%, to a mere 100,000,000; but after some years of adaptation, most rabbits survived infection, and the rabbit population rebounded. While on the one hand the rabbits were obviously selected for resistance to myxomavirus,³ the virus also did in fact evolve to reduced virulence. This was shown in a classic study by Fenner and Marshall in 1957.⁴ In this unusual situation, they still had samples both of the original virus, and of a non-evolved rabbit population in Europe, so they could do direct comparisons. The new strains of the virus circulating in Australia were less lethal. What’s more, those rabbits that did die, took much longer to do so, surviving for several weeks instead of 5 days or so as with the original highly lethal strain.But Fenner’s work also showed why this isn’t a general law, and showed one of the problems with extrapolating this to the extreme of avirulence — because in fact the virus did not evolve to avirulence, it evolved to moderate virulence and then stayed there, killing about 50% of the rabbits it infected. Fenner & Marshall said: “The overall trend towards moderate virulence (grade III) … can be explained by the selective advantage for mosquito transmission of strains which cause extensive and long-persisting infectious skin lesions in rabbits.”In other words:⁵

The less-virulent virus took 3 to 4 weeks to kill a rabbit instead of 6 to 10 days, so that sick rabbits could be bitten by mosquitoes and fleas for 3 to 5 times as long as a rabbit suffering from the highly virulent strain. The milder strain was therefore more successful in infecting rabbits, and it spread rapidly. Through this selection the virus evolved to a less-virulent form.

(The map at right is from a later paper by Fenner,⁶ showing a similar phenomenon in British myxomavirus. Note that most of the strains isolated here, a decade or so into enzootic myxomavirus, are Grade III, “moderate”, killing “just” 70-90% of infected rabbits, rather than the relatively avirulent grade V or the brutally lethal grade I that was the original infection.)This highlights a key for this sort of evolution to work. There needs to be a direct link between increased transmission of the disease, and reduced virulence. The issue of a new supply of hosts, which is what most people seem to think is the critical factor, seems to be relatively minor. Conversely, if there’s a link between increased transmission and increased virulence, then the balance will not favour the pathogen becoming benign. If, for example, you are a virus that spreads by causing your host’s blood to explode out of its body, or if you destroy your host’s brain and force it to run about furiously biting anything in sight, or if you are spread through insect vectors that find your host an easier target when it’s moribund — then becoming less lethal is unlikely to help you. This has been proposed in detail, and to some extent experimentally tested, most prominently by Paul Ewald.⁷ I don’t know enough about the evolutionary and epidemiological sides to comment intelligently,⁸ so I’ll stop here, but with this quote from Ewald that explains why this sort of theoretical work can be important:⁹

Insights into the evolution of virulence may aid efforts to control or even prevent emerging diseases. Specifically, dangerous pathogens can be distinguished from those that pose relatively little threat by identifying characteristics that favor intense exploitation of hosts by pathogens, hence causing high virulence. Studies to date have implicated several such characteristics, including transmission by vectors, attendants, water, and durable propagules.

1. Also, as is usually the source for whatever I’m blathering about here, it’s something I ran across in my reading anyway, and this is one way I help solidify things in my mind.[↩]
2. Everyone who is anyone, at least[↩]
3. A major complication in interpreting this sort of phenomenon — if you don’t have an original population of the hosts, how can you tell if it was the pathogen or the host that evolved?[↩]
4. A comparison of the virulence for European rabbits (Oryctolagus cuniculus) of strains of myxoma virus recovered in the field in Australia, Europe and America. Fenner F, Marshall ID. J Hyg (Lond). 1957 Jun;55(2):149-91. [↩]
5. From a commentary on rabbit calicivirus at the Australian Academy of Sciences’ Nova page[↩]
6. Evolutionary Changes In Myxoma Virus In Britain. An Examination Of 222 Naturally Occurring Strains Obtained From 80 Counties During The Period October-November 1962. Fenner F, Chapple PJ. J Hyg (Lond). 1965 Jun;63:175-85. [↩]
7. For example, Pathogen survival in the external environment and the evolution of virulence. Walther BA, Ewald PW.. Biol Rev Camb Philos Soc. 2004 Nov;79(4):849-69.) [↩]
8. Not that that usually stops me[↩]
9. The evolution of virulence and emerging diseases. Ewald PW. J Urban Health. 1998 Sep;75(3):480-91.[↩]