Mystery Rays from Outer Space

Inbreeding is a bad thing, genetically, and almost all species have ways of avoiding it. One way of avoiding inbreeding is to recognize individuals who are related to you, and not mate with them. That’s not so difficult when you’re a big-brained, highly social animal like a wolf, or a human, who have lots of brain devoted to issues of who is who and where they stand in a group. It’s a little more challenging for mice, though.

How do mice distinguish individuals? How do they determine relatedness? For the past 30 years¹, the answer has been MHC: Mice select mates that differ at the major histocompatibility complex.

When I talked about this last fall, in the context of MHC diversity, I was rather skeptical that mating preference was the major driver of MHC diversity, quoting Piertney and Oliver:²

A lack of repeatability of several studies, and an apparent plasticity in response across experiments, questioned the robustness of the data, and the general relevance of mate choice as a primary driver of MHC diversity.

It didn’t occur to me to question the fundamental observation that MHC is even involved in distinguishing relatedness.

MHC seemed like a logical candidate for distinguishing individuals and determining relatedness because of its great polymorphism: in an outbred population, the MHC is so variable that few individuals are identical across the region, and individuals with similar MHC are most likely related to some extent. ³ And in fact, mice clearly can distinguish differences in MHC type by smell. ⁴ However, that doesn’t mean that mice recognize different individuals, or determine relatedness, by the differences in MHC. A couple of papers from Jane Hurst’s group in the past year suggest that in fact they do not. ^5,6

Hurst’s group tried to move away from the artificial situation of highly-inbred lab mice, using instead wild mice breeding in semi-natural conditions. They find that under these conditions, mice do (as expected) avoid breeding with close relatives. But this incest avoidance doesn’t correlate with MHC type. Instead, there was a strong correlation with MUP type.

What, you cry, is MUP? These are “major urinary proteins”, which are known to be highly polymorphic in wild mouse populations — though not in lab mice — and which are also known to be very important in scent marking. Indeed, the only known function of MUPs is in scent marking. The lack of variability of MUPs in lab mice might have led to the use of MHC as markers instead in those studies, but in Hurst’s study MHC didn’t contribute to incest avoidance:

By contrast, MUP sharing had a strong and highly significant effect on the likelihood of successful mating (Table 1: model 3, p = 0.005; Figure S1). Specifically, there was no deficit when only one MUP haplotype was shared, but there were many fewer matings between mice that shared both MUP haplotypes (complete match) than expected under random mating conditions (Table 1: model 4, p < 0.002). … Mice thus avoid mating when shared MUP type reliably indicates very close relatedness.

Incidentally, this is consistent with a recent paper from Peter Overath and Hans-Georg Rammensee.⁷ They looked for influences on urine odor in mice (try writing to your Mom and tell her that’s what you’re doing for your living, by the way, and see how long it takes before she starts talking about your cousin the investment banker) and didn’t find any influence of MHC:

… within the limits of the ensemble of components analysed, the results do not support the notion that functional MHC class I molecules influence the urinary volatile composition.

(However, there are non-volatile as well as volatile components to urine odor, so this isn’t definitive.)

MUPs are highly polymorphic in wild domestic mice, but are non-polymorphic (actually, basically non-existent) in humans. (In fact, MUPs are non-polymorphic even in Mus macedonicus, a mouse species closely related to M. musculus domesticus, but a species that doesn’t need as careful management of increeding because individuals normally disperse more. ) That means that MUPs can’t be a universal mechanism for inbreeding avoidance, so the work on MHC-linked mate choice in other species might still be valid. However, I still think the work on MHC and mate selection in humans is mostly pretty crappy unconvincing. Since the work in humans leans heavily on the assumption that MHC is important in mate selection in mice, that work can be looked at with an even more jaundiced eye now, I think.

1. Yamazaki, K., Boyse, E. A., Mike, V., Thaler, H. T., Mathieson, B. J., Abbott, J., Boyse, J., Zayas, Z. A., and Thomas, L. (1976). Control of mating preferences in mice by genes in the major histocompatibility complex. J Exp Med 144, 1324-1335[↩]
2. Piertney, S. B., and Oliver, M. K. (2006). The evolutionary ecology of the major histocompatibility complex. Heredity 96, 7-21.[↩]
3. A review is here: Adv Genet. 2007;59:129-45. Genetic basis for MHC-dependent mate choice. Yamazaki K, Beauchamp GK.[↩]
4. For example, Carroll, L.S., Penn, D.J., and Potts, W.K. (2002). Discrimination of MHC-derived odors by untrained mice is consistent with divergence in peptide-binding region residues. Proc. Natl. Acad. Sci. USA 99, 2187–2192.[↩]
5. Sherborne, A., Thom, M., Paterson, S., Jury, F., Ollier, W., Stockley, P., Beynon, R., Hurst, J. (2007). The Genetic Basis of Inbreeding Avoidance in House Mice. Current Biology, 17(23), 2061-2066. DOI: 10.1016/j.cub.2007.10.041[↩]
6. The Genetic Basis of Inbreeding Avoidance in House Mice
   Amy L. Sherborne, Michael D. Thom, Steve Paterson, Francine Jury, William E.R.
   Ollier, Paula Stockley, Robert J. Beynon, and Jane L. Hurst. Curr Biol. 2007 December 04; 17(23): 2061–2066. doi: 10.1016/j.cub.2007.10.041 [↩]
7. Röck F, Hadeler K-P, Rammensee H-G, Overath P (2007) Quantitative Analysis of Mouse Urine Volatiles: In Search of MHC-Dependent Differences. PLoS ONE 2(5): e429. doi:10.1371/journal.pone.0000429.[↩]

I just got back from Toronto, where I visited the Darwin exhibit at the ROM;¹ the picture above is what my kids look like after being sternly told to stop goofing around.²

Bonus pictures! while I stall on putting up a real post here:

Cap’n Matthew navigates the HMS Beagle through the stormy seas of the ROM	Alex the Albertosaurus

1. Also made it to a ballgame to see my Red Sox lose to the Jays, and the Ontario Science Center for the kids[↩]
2. Yeah, no pictures allowed at the Darwin exhibit. They didn’t shoot me, though.[↩]

I’ve observed before that the common belief that viruses evolve toward avirulence is not particularly true. It’s more accurate to say that viruses evolve toward improved transmission. Some viruses are better transmitted if they let their host survive longer, but other viruses have to be virulent in order to spread. The former may evolve toward reduced (though not necessarily loss of) virulence, but the latter would “want” to maintain stable virulence.

What about increasing viral virulence? What could drive that?

There’s at least one fairly well-documented example of that. The increase in virulence is probably because of a change in the virus’s environment that  forces the virus to become more virulent in order to continue to transmit efficiently. Ironically, the environmental change is vaccination.

As far as I know — I want to put this up front, to forestall the vaccine loons — there’s no instance where this has happened with a vaccine used for humans. ¹ I’m talking about a chicken vaccine, for Marek’s Disease.

Marek’s Disease Virus (MDV) is an extraordinarily interesting virus. It’s a herpesvirus of chickens; it causes, among other symptoms, tumors. MDV was a relatively minor problem when chicken farming was a backyard industry. When very large, intensive commercial chicken farms arose, the virus was able to sweep through flocks and cause truly enormous losses. The first Marek’s Disease vaccine, introduced in the 1960s, reduced losses by some 99%. (Incidentally, this was the first vaccine ever to prevent cancer.)

But the 99% protection rate didn’t last long. Losses began to creep up once again, as more virulent viruses arose. New vaccines have been introduced a couple times; each time losses dropped, but then once again new and increasingly-virulent viruses arose. Marek’s Disease viruses isolated today are far more virulent than the relatively benign viruses of the 1960s and early 1970s; the original vaccine is essentially useless against them.

The figure at right² (click for a larger version) shows the virulence of virus strains isolated over a ten-year period — although there’s a lot of variability, there’s a pretty clear upward trend. (This chart — and all the others I could find — only shows changes relatively late in the story, skipping the interesting periods in the 1970s and early 1980s when the first changes in virulence were noted. I think this is a technical issue of having the appropriate strains available for comparison. However, see: Increased virulence of Marek’s disease virus field isolates. Witter RL. Avian Dis. 1997 Jan-Mar;41(1):149-63. doi:10.1016/j.tvjl.2004.05.009 for a more detailed analysis of MDV strain virulence over the years.)

This evolution is actually very reminiscent of the myxoma/rabbit co-evolution story I’ve talked about, here and here. Australian rabbits have evolved to become much more resistant to myxoma virus than their European cousins. In this case, MDV is more analogous to the rabbits than to myxoma — evolving mechanisms to persist and replicate in the face of a lethal challenge (for the rabbits, myxoma virus; for Marek’s Disease virus, the vaccine-derived immunity).

Before rabbits could evolve resistance, there had to be some survivors of myxoma infection. In that case, myxoma virus itself evolved to become somewhat less virulent (70-90% lethal, instead of 98%). In the Marek’s Disease story, a key factor is that the vaccines all suck³ in their ability to actually prevent infection; they prevent the disease, but viruses can still infect vaccinated birds, although the virus replicates slower (which reduces transmission).

This is a recipe for virulence. Viruses in general evolve toward improved transmission. The MDV vaccine reduces, but doesn’t eliminate, transmission. Increasing replication in the face of the vaccine increases transmission. Increasing viral replication also increases viral virulence.⁴

This probably isn’t the whole story (there’s some evidence that the virus was already evolving toward increased virulence even before the vaccine was introduced — perhaps related to changes in its environment brought about by factory farming), and the mechanisms underlying the changes in virulence are not known, but the solution would seem to be clear: Develop a Marek’s Disease vaccine that will induce sterilizing immunity, as do most vaccines used against human viruses. That way, there’s no survivor virus that can act as a seed for evolution of virulence.

Unfortunately, of course, herpesviruses like MDV are notoriously difficult to vaccinate against. There’s still no commercial vaccine against herpes simplex virus, in spite of decades of research. Feline herpesvirus vaccine, which is universally used among pet cats, is like Marek’s in that it prevents symptoms but doesn’t prevent infection. (There is an effective vaccine against varicella-zoster virus [chicken pox] which does seem to effectively prevent infection — an exception to the rules.) So the chicken world is forced to stick with the non-sterilizing vaccines, even though “MD vaccines also appear to have a malign influence on the continued evolution of the pathogen itself.” ²

1. I’m not saying there’s no such instance, but I don’t know of one.[↩]
2. Nair, V. (2004). Evolution of Marek’s disease — a paradigm for incessant race between the pathogen and the host. The Veterinary Journal DOI: 10.1016/j.tvjl.2004.05.009[↩][↩]
3. Note rigorous technical terminology[↩]
4. This is not a universal equation; virus virulence isn’t necessarily linked to increased replication, for example.[↩]

Some leaf-cutter ant lineages are more likely to become queens than other lineages; they “cheat”. These lineages are a minority, about 20%, of all leaf-cutter lineages. I’m fine with all that. What puzzles me is this quote:

“The rarity of the royal lines is actually an evolutionary strategy by the cheats to escape suppression by the altruistic masses that they exploit.”

Bill Hughes, quoted in Science Daily News. It’s not a misquote, either; the abstract of the paper in question¹ says essentially the same thing:

The rarity of royal cheats is best explained as an evolutionary strategy to avoid suppression by cooperative genotypes, the efficiency of which is frequency-dependent.

What am I missing here? The strategy is successful because it’s rare, sure. Is he arguing that there is positive selection for rarity, as opposed to a strategy that is selected for when it’s rare, and selected against when it’s common?

1. Proc. Natl. Acad. Sci. USA doi:10.1073/pnas.0710262105
   Genetic royal cheats in leaf-cutting ant societies
   William O. H. Hughes, and Jacobus J. Boomsma [↩]

The recent papers on TRIM5-cyclophilin fusion proteins as retroviral restriction systems,¹ and my nattering about myxomavirus the other day, reminded me of another paper I recently read.

TRIM5 is one of a family of proteins that prevent replication of certain retroviruses; the TRIM5-CYP fusion protein protects against infection so that, for example, feline immunodeficiency virus doesn’t effectively infect rhesus macaque cells. This is a specific instance of a general problem: Why are are some viruses only able to infect certain species, while others leap around between species like the chamois of the alps? This isn’t just relevant to retroviruses. Emerging virus infections are an ongoing problem, and lots of the emerging viruses are jumping into humans from some other species — HIV, Ebola, and SARS being particularly obvious examples out of many.

In many cases, of course, viruses are specifically adapted to their receptors, and another species may not have the precise receptor, or the precise shape or sequence the virus needs to get in a cell in the first place. Similarly, the virus may depend on some species-specific structure for some step in replication. But there are viruses that can cheerfully replicate in insects, birds, and mammals; fundamental replication equipment is pretty conserved.

In other cases — like the TRIM5 story — hosts may have evolved systems to restrict viruses, and only those viruses that were resistant, or that developed resistance, to the restriction systems were able to persist. In this case, host restriction is the flip side of viral immune evasion;² viruses that are unable to counteract the host’s immune adaptations go extinct in that host. Most such extinctions are invisible; species-specific viruses are (at least in some cases) the fossil evidence for ancient viruses.

Beta-herpesviruses — cytomegaloviruses, for example — are among the most species-specific virus families. (I’m approaching my point here.) Broadly speaking, the way you get a new beta-herpesvirus is when its host speciates. (The same is generally true for other herpesvirus families, but there’s more evidence for cross-species jumping of some of the other families.) How come? What is it that’s restricting mouse cytomegalovirus (MCMV) to mice, and prevents them from infecting humans? The answer was a bit of a surprise to me, actually: Apoptosis is a major factor.

If you’d asked me a couple of years ago, I’d have guess that MCMV would simply never get in to human cells — wrong receptor, I’d have suggested. I would have been wrong.³ Jurak and Brune (and here I have reached my point) showed a couple of years ago that MCMV gets into human cells just fine, ⁴ but in most cases it’s not able to do anything afterward; it doesn’t spread to neighboring cells, and eventually it just fades away from the culture.

The exceptions, as is often the case, were interesting. In two of the cell lines they tested, MCMV not only entered, but replicated and spread (though a thousand times less well than in permissive mouse cells). Those cell lines are 293 and 911 cells, and right now the virologists⁵ in my audience⁵ are nodding and saying, “Aha!” — because 293 and 911 cells have something in common: They both contain chunks of a human adenovirus genome.⁶ There are a bunch of functions included in those chunks of genomes, but to cut to the chase, it was the anti-apoptosis function that proved to be important here.

Human cells infected with MCMV undergo apoptosis, dying before the virus can replicate. Swapping various anti-apoptotic genes into human cells renders them sensitive to MCMV infection.

Blocking apoptosis is a very common, if not universal, function of viruses, so clearly apoptosis is a hugely important antiviral process. Paradoxically, that’s why I was surprised to find out that apoptosis limited MCMV replication in human cells. The apoptosis pathway is pretty conserved, and most of the viral anti-apoptotic molecules I could name off the top of my head^7,8 act on many species. (The baculovirus protein IAP (”Inhibitor of APoptosis”) — evolved to function in cells of infected insects — works just fine in mammalian cells,⁹ for example.) It’s not at all clear to me why MCMV, which can presumably inhibit apoptosis in mouse cells, can’t do so in human cells. Human CMV does block apoptosis in human cells, through more than one pathway, and swapping the HCMV gene into MCMV makes the mouse virus able to replicate in human cells. Presumably the MCMV gene(s) that have the similar function are tuned to the mouse version of the apoptosis pathway.

Incidentally, since apoptosis is an anti-tumor mechanisms as well as an antiviral, many tumors have down-regulated components of their apoptosis pathways. This, at least potentially, makes tumors targets for viruses that can only grow where apoptosis is inhibited, and this is an active area of research for the various oncolytic viruses. As well MCMV (which probably isn’t a good candidate for oncolysis, because it replicates poorly in human cells even when apoptosis is blocked) our old friend myxomavirus is at least partly restricted by apoptosis; knocking out the myxoma M-T5 protein, an anti-apoptosis gene, eliminates its pathogenicity altogether in rabbits, “making MV an excellent oncolytic candidate”. ¹⁰

I don’t want to leave the impression that apoptosis is the usual reason for host restriction. There are all kinds of other explanations. TRIM family proteins, as I said, limit retroviruses, as do APOBEC family proteins; SARS adapted to humans by (among other things) mutating its receptor to bind better to human angiotensin-converting enzyme 2; ¹¹ myxomavirus are apparently restricted to some species because their immune response modifiers are species-specific¹² (and I will probably talk more about that some time); and so on.

1. Virgen CA, Kratovac Z, Bieniasz PD, Hatziioannou T (March 4, 2008) Independent genesis of chimeric TRIM5-cyclophilin proteins in two primate species. Proceedings of the National Academy of Sciences 105.:3563-3568 10.1073/pnas.0709258105
   Wilson SJ, Webb BLJ, Ylinen LMJ, Verschoor E, Heeney JL, et al. (March 4, 2008) Independent evolution of an antiviral TRIMCyp in rhesus macaques. Proceedings of the National Academy of Sciences 105.:3557-3562 10.1073/pnas.0709003105
   Brennan G, Kozyrev Y, Hu S (March 4, 2008) From the Cover: TRIMCyp expression in Old World primates Macaca nemestrina and Macaca fascicularis. Proceedings of the National Academy of Sciences 105.:3569-3574 10.1073/pnas.0709511105[↩]
2. For rather generous uses of “immune evasion”[↩]
3. And should have known better, because it was shown 35 years ago that MCMV can enter humans cells: Kim KS , Carp RI (1972) Abortive infection of human diploid cells by murine cytomegalovirus. Infect Immun 6: 793-797 [↩]
4. Jurak, I., Brune, W. (2006). Induction of apoptosis limits cytomegalovirus cross-species infection. The EMBO Journal, 25(11), 2634-2642. DOI: 10.1038/sj.emboj.7601133[↩]
5. If any[↩][↩]
6. For 193 cells: Graham FL , Smiley J , Russell WC , Nairn R (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36: 59-74
   For 911 cells:
   Fallaux FJ , Kranenburg O , Cramer SJ , Houweling A , van Ormondt H , Hoeben RC , van der Eb AJ (1996) Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum Gene Ther 7: 215-222 [↩]
7. A fun ice-breaker for your next cocktail party — name that viral apoptosis inhibitor![↩]
8. And this is why I don’t get invited to many cocktail parties[↩]
9. Duckett CS, Nava VE, Gedrich RW, Clem RJ, Van Dongen JL, et al. (June 3, 1996) A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors. EMBO J 15.:2685-94.[↩]
10. Werden SJ, McFadden G (January 2008) The role of cell signaling in poxvirus tropism: The case of the M-T5 host range protein of myxoma virus. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 1784.:228-237 doi:10.1016/j.bbapap.2007.08.001[↩]
11. Li W, Zhang C, Sui J, Kuhn JH, Moore MJ, et al. (April 20, 2005) Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J 24.:1634-43.
    Sheahan T, Rockx B, Donaldson E, Sims A, Pickles R, et al. (March 2008) Mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium. J Virol 82.:2274-85.[↩]
12. Wang F , Ma Y , Barrett JW , Gao X , Loh J , Barton E , Virgin HW , McFadden G (2004) Disruption of Erk-dependent type I interferon induction breaks the myxoma virus species barrier. Nat Immunol 5: 1266–1274[↩]

I’ve previously talked about the common misconception that viruses evolve toward benignity. This is usually phrased something like, “Natural selection favours viruses with low pathogenicity/virulence (so they don’t eradicate their hosts)“, or “Viral pathogenesis is an abnormal situation of no value to the virus“. This claim is clearly wrong — “clearly” both through common sense, and through observation.

Common sense tells us that pathogenicity does benefit at least some viruses. If a virus is spread through fecal/oral contamination (like noroviruses, say), then increasing fecal production will increase viral spread; we perceive that as diarrhea, fluid loss, illness and perhaps death — pathogenicity. In this case, the virus obviously benefits from virulence; there’s no pressure on the virus to evolve toward reduced pathogenicity, because that would reduce its transmission.

I talked about this with myxomavirus — one of the few well-studied cases where the concept can actually be tested. Myxoma was introduced to Australia 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. However, the virus did not evolve to “low pathogenicity”; rather it evolved to a point where it killed about 70% of rabbits it infected. (To put that in context, it’s the same ballpark as smallpox, or Ebola — viruses which are not usually considered to have low virulence.) In this case, the reduced (but still very high) pathogenicity correlated with a longer period of transmission; the rabbits were sick for weeks, and less able to fend off the mosquitos and fleas that spread the virus. The most virulent virus killed the rabbits so fast that not many mosquitos got a chance to bite them. Hence, evolution to reduced, but still very severe, virulence.

But there’s another side of the equation. The host would, very obviously, benefit from reduced viral pathogenicity. This is something that’s hard to separate in natural experiments. When we see, for example, deer mice surviving with minimal disease while infected with Sin Nombre virus, how can we tell if the virus has adapted to the host, the host to the virus, or both?

In this natural myxomavirus experiment, we can in fact address that question, because Australian rabbits had to deal with the virus while their European cousins didn’t. And as you’d expect, Australian rabbits are actually relatively resistant to myxomavirus.

Below are data from Fenner¹ showing survival of wild Australian rabbits when exposed to the same strain of myxomavirus (click for a larger version). (Because the virus was deliberately introduced, it was known what the original strain was, and new strains with defined virulence in European rabbits could be isolated and saved.) The Y axis is the percent of rabbits responding with a certain level of disease; the X axis shows the number of epidemics that had spread through the country before sampling the rabbits.

In the first couple epidemics rabbits were almost all severely infected; but after selection by two or three epidemics, many of the rabbits are only moderately affected by the same strain of virus that killed almost all of their ancestors, and after six or seven epizootics, most rabbits survived and many were only mildly sick:

The belief that evolution is a slow process, undetectable by our mayfly eyes, is long obsolete. Here’s one example of how fast changes can become fixed in a population. As Fenner said:

it was then thought that it might be decades before resistance became evident, and that challenge with something less than fully virulent virus would make it easier to observe the first changes. To our surprise, within a few years, in a population of’ rabbits that had been exposed annually to myxomatosis, the case fatality rate fell from about 90% to about 50%, and eventually even lower than that.

But it’s not so simple. For example, why did it take a few epidemics before resistance arose in the rabbits?

Probably the reduced viral virulence played an important part in that. With a 99.8% mortality, there’s not much room for resistance to arise. After the virus settled down and spared 30% of its hosts, there was room for survivors to pass on their resistance genes. But there are limits:

… the rate of acquisition of resistance had reached a plateau after about six generations of selection (W. R. Sobey, personal communication, 1982). Perhaps this represents a limit to what can be achieved by selection of the pre-existing genotypes; any further change may require the occurrence and selection of mutations for resistance.

(That was written in 1983, and I don’t actually know if resistance has progressed further.)

We don’t know (or at least I don’t know; and I haven’t seen anything published on it) exactly what the resistance factors are in the resistant rabbits. However, it’s interesting that apparently a major cause of myxomavirus disease is “an acute and overwhelming immunopathological response to the virus“,² because this brings me back to the deer mouse/hantavirus story I mentioned earlier. It seems that mice respond to Sin Nombre virus with a regulatory T cell response — dampening immune responses rather than enhancing them — and this may be one reason they don’t show disease (i.e. they’ve removed the immunopathological side of the equation). I wonder if Australian rabbits have also evolved a tendency to develop TReg responses to myxoma virus.

In any case I can’t offer a better summary than Fenner’s:

Observations of its effects after some 30 years show that a fantastically virulent virus did not eradicate the rabbit, but that selection for transmissibility, especially during the off-season (winter in Australia) allowed the early emergence and rapid dominance of strains of lowered virulence. We now see a fascinating interplay between genetic changes in host and virus.

1. Fenner F. Biological control, as exemplified by smallpox eradication and myxomatosis. Proceeding of the Royal Society of London, Series B, The Florey Lecture, 1983: Biological Control, as Exemplified by Smallpox Eradication and Myxomatosis 1983;218:259-285.[↩]
2. Silvers L, Inglis B, Labudovic A, Janssens P, van Leeuwen B, et al. (April 25, 2006) Virulence and pathogenesis of the MSW and MSD strains of Californian myxoma virus in European rabbits with genetic resistance to myxomatosis compared to rabbits with no genetic resistance. Virology 348.:72-83 doi:10.1016/j.virol.2005.12.007[↩]

Last week I talked about the evolution of noroviruses, which have repeatedly thrown out new strains and caused new epidemics over the past 20-odd years. In contrast — and rather unexpectedly, at least for me — it seems that HIV is not changing its virulence over time; at least, not in North America.

You can think of reasons why HIV “should have” either increased or decreased its virulence since entering North America. For example, HIV entered the human population relatively recently; perhaps it’s still adapting to its new host, and is learning how to more efficiently infect us. On the other hand, it’s widely (though incorrectly) believed that pathogens inevitably evolve toward reduced virulence; perhaps HIV follows this path as well. (Similarly, at least in theory, the human population could be adapting to the virus, something that certainly happens with other species and viruses. However, 30 years or so seems pretty short for this kind of evolution in humans.) The HIV epidemic in North America started in the early 1980s, which has given the virus lots of time to evolve one way or another (compare to noroviruses, which have thrown out six epidemic-causing variants¹ since 1995).

Experimental evidence has pointed in all directions — some suggests that HIV is becoming less virulent, some that it is becoming more virulent, and some says it’s staying the same. There are all sorts of complications in measuring this. What exactly is “virulence“, anyway — time to death? Death rate? Rate of replication? Transmission? In the absence of a suitable animal model the most direct answers can’t really be tested directly. What’s more, because of the vast improvement in anti-retroviral treatment over the years, it’s hard to compare clinical course. And the virus is intrinsically so variable that you’d need large numbers just to be sure you’re analyzing a real trend.

A recent paper in PLoS ONE² tried to resolve the question by looking at clinical correlates of virulence, and they reached the conclusion that the virus hasn’t changed significantly (in terms of virulence, by this definition) over time:

We tested for associations between calendar year of seroconversion and three prognostic markers of disease progression in the MACS cohort between 1984 and 2005. Our results showed no significant trends in set point plasma viral RNA load, CD4 cell count after seroconversion, or the rate of CD4 cell decline in the first three years after seroconversion. Moreover, estimates of change in these markers over time were very close to zero. Thus, the results of this study do not support the hypothesis that there has been any important change in the virulence of HIV-1 over this time period in this cohort.

Is this study likely to end the controversy? I really doubt it; there are just too many limitations in this kind of study. (I do think, though, that it helps establish bounds: it’s unlikely that HIV virulence has changed massively either way since it arrived in North America.) Nor does it help us going forward: HIV strains with different virulence may be arising even now. Still, it’s an interesting finding. My own prejudice would have been that HIV virulence would be increasing, but I’m happy to set that aside for now.

1. Simplified![↩]
2. Herbeck, J.T., Gottlieb, G.S., Li, X., Hu, Z., Detels, R., Phair, J., Rinaldo, C., Jacobson, L.P., Margolick, J.B., Mullins, J.I., Tripathy, S. (2008). Lack of Evidence for Changing Virulence of HIV-1 in North America. PLoS ONE, 3(2), e1525. DOI: 10.1371/journal.pone.0001525[↩]

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[↩]

What with visiting speakers and new faculty recruitment, I’ve been out late every night this week; what with committee meetings¹ and trying to squeeze in experiments, I’ve been up early every morning; and what with teaching starting up again, seminars from visiting speakers and recruitees, and faculty meetings, I’ve had little time for other stuff. So this is going to be a short post. ²

A while ago I talked about lamprey immune systems. The key points are that lampreys …

• have an immune system
• that works pretty well
• and in concept looks a lot like our immune system, with lymphocytes and specific receptors
• but the receptors are utterly unlike our T cell receptors and antibodies
• using instead of the immunoglobulin domain structure, a leucine-rich repeat (LRR) structure;
• and, instead of using RAG-based recombination, uses a gene conversion system to generate diversity.

There’s a temptation, even for those who intellectually know better, to assume that “primitive” animals have “worse” systems; so because lampreys are more like the common ancestor of vertebrates, their immune system must be “worse”. (Hagfish and lampreys, which may have diverged some some 500 million years ago — see the figure to the right;³ click for a larger version — have very similar immune systems, so this system must be at least that old.) In some ways the mammalian immune response does seem to have some advantages — faster memory response, for example. Still, lamprey immune systems have served them well for 500 million years, which is more than we can say about ours; and in some other ways lampreys do better than we do. They have if anything a greater diversity to their receptors, for example, potentially generating more than 10¹⁴ different receptors — compare to our roughly 10⁸ T cell receptors.

Max Cooper, who has done much of the work on lamprey immunity,⁴ has just published a paper showing off some other unusual properties of lamprey immune receptors. ⁵ Since there’s no system for making lamprey monoclonal antibodies that’s analogous to the mouse monoclonal antibody systems, he used a molecular cloning approach to express monoclonal variable lymphocyte receptor (VLR) -B cDNAs from immunized lampreys.

What did they get?

They got soluble “antibodies”, capable of the highly specific recognition that’s seen in conventional monoclonal antibodies. The VLR-B antibodies are extraordinarily stable, maintaining binding at pH 1.5 and maintaining structure at pH 11, as well as after incubation at 56 ^oC for a couple days or at room temperature for weeks. ⁶ Although the individual LRR subunits have relatively low binding affinity, they are secreted as multimers of eight to ten subunits (see the diagram to the left), and as a result the VLR-B binding ability can be at least as good as mouse monoclonals: “Equal concentrations of VLR4 and EA2-1, starting at 0.5 mg/ml, were serially diluted in 10-fold increments and scored for the degree of spore agglutination. Spore agglutination by VLR4 was detected at a concentration 1,000-fold more dilute (5 pg/ml) than the mouse monoclonal antibody (5 ng/ml).”

Finally, as opposed to mouse monoclonals, these are single proteins; conventional mouse monoclonals have two components, a heavy and a light chain. That makes VLR-B easier to work with in some ways: “The single peptide composition of VLR-B antibodies makes them more amenable to molecular engineering, including manipulation of the antigen binding site by mutagenesis and fusions to the coding sequences of other peptides, such as enzymes, toxins, and epitope tags to extend their functional capabilities.” ⁷

These things clearly have potential to be useful in all kinds of things — a nice example of basic research giving rise to clinically and commercially useful tools.

1. Proof, if proof were needed, that deans are evil: 7:30 AM meeting with the dean[↩]
2. Also, I just realized I really, really need to get some flowers for my wife today, don’t I.[↩]
3. From: Modern look for ancient lamprey. Philippe Janvier. Nature 443, 921-924(26 October 2006 [↩]
4. It was his talk at the Autumn Immunology Conference in Chicago a couple of years ago that made me realize how fascinating the subject is[↩]
5. Herrin, B.R., Alder, M.N., Roux, K.H., Sina, C., Ehrhardt, G.R., Boydston, J.A., Turnbough, C.L., Cooper, M.D. (2008). Structure and specificity of lamprey monoclonal antibodies. Proceedings of the National Academy of Sciences, 105(6), 2040-2045. DOI: 10.1073/pnas.0711619105[↩]
6. Conventional antibodies are pretty stable, but not up to this level.[↩]
7. I think it’s camels that make single-chain antibodies, and there’s been interest in developing monoclonal systems based on camel antibodies for the same reason.[↩]

A while ago I talked about evolution of the herpesviruses, and I said:

We know of 200-odd herpesviruses so far, and more are being identified practically daily. It’s likely that virtually every animal species has its own set of unique herpesviruses. This is probably because herpesviruses are very host-restricted (rarely infecting more than a single species) and set up latent, life-long infection. When an animal population speciates, its complement of herpesviruses speciates along with it.

Word of my blog post spread like wildfire (as is inevitable for a blog that is read by upward of five people, including my mother) and Duncan McGeoch hastened to correct me with a fascinating paper now in pre-print form at the Journal of Virology:
Ehlers, B., Dural, G., Yasmum, N., Lembo, T., de Thoisy, B., Ryser-Degiorgis, M., Ulrich, R.G., McGeoch, D.J. (2008). Novel mammalian herpesviruses and lineages within the Gammaherpesvirinae: Cospeciation and interspecies transfer. Journal of Virology DOI: 10.1128/JVI.02646-07

McGeoch’s group went herpesvirus hunting (”Be