Mystery Rays from Outer Space

About 15 minutes after I wrote my last article on influenza variation, I was reading the Journal of Virology  and ran across another paper¹ on the same thing, that at least partly addresses some of the missing points in the earlier ones.

To brutally truncate my earlier comments: influenza should generate a huge number of mutants as it replicates; but in the few studies that have been done, not all that many variants have actually been detected.

One of the points I raised was that the influenza variation was sampled at the end-point of the infection — after the patient had died, in the paper I talked about the other day.² Even though the virus had been through the maximum number of replication cycles, it had also experienced the maximal selection pressure, potentially reducing the number of surviving mutants. Is it possible that more variants arose earlier in the infection, but died off before they were detected?

This new paper¹  actually looked at exactly that. They used canine influenza as their model, so they could deliberately infect their patients and track through the infections from the beginning through the end. Even though they used a technique that is much less sensitive to mutations (and is probably more error-prone as well) they found tons of variation, and the pattern they found is fascinating:

Mutations arose readily in the infected animals and reached high frequencies in some vaccinated dogs, but they were mostly transient and often were not detected on subsequent days. Hence, CIV populations are highly dynamic and characterized by a rapid turnover of likely deleterious mutations. ((Hoelzer, K., Murcia, P., Baillie, G., Wood, J., Metzger, S., Osterrieder, N., Dubovi, E., Holmes, E., & Parrish, C. (2010). Intrahost Evolutionary Dynamics of Canine Influenza Virus in Naive and Partially Immune Dogs Journal of Virology, 84 (10), 5329-5335 DOI: 10.1128/JVI.02469-09))

(My emphasis) This (assuming it holds true in other studies) beautifully resolves much of the difference between the expected level of variation, and the level that’s observed at any one time point of infection. The explanation is that the variation does indeed appear, but it doesn’t persist.  There is variation is over time as well as at any one time point.

Figure 1. Variation between challenge influenza virus (yellow) and virus isolated from two naïve dogs 2 to 4 days after infection 1

There are a lot of very cool things about this study that I’m not going to talk about (differences between vaccinated and unvaccinated animals, evidence for antigenic escape) but there are two things that I thought were particularly exciting.

First is the question of why the mutations seem to be so transient. Part of that could just be chance, part of it is probably selection against deleterious mutants.

But it’s also worth keeping in mind that the viruses are replicating in a dynamic, rapidly-changing environment. The virus enters a host whose immune system is at rest but that immediately recognizes viral infection and ramps up interferons,  then other cytokines, then innate antiviral systems that build up and spill over into an adaptive immune response  …  a whole range of inflammation whose mediators and effectors change from hour to hour. Is this changing environment selecting for mutations that are briefly beneficial, and that then become deleterious as the situation changes a few hours later?

Second – when we think about viruses that are able to jump from one species to another, we think usually of mutants, virus that may be less fit in their “proper” hosts but adequately fit in some other species. (In fact canine influenza itself is a great example of this, a virus that jumped from horses into dogs six or seven years ago.  It is essentially equine influenza, but compared to the equine version it has a half-dozen variants that make it more suitable for replication in dogs.)

If we look at any particular time point we may not find any of these potential emergent mutants. But if we look at all the time points, as in this study, perhaps these potential species-jumping mutants are popping up all the time, but only for a few hours at a time:

This observation suggests that mutations that facilitate adaptation to a new host species might occur transiently in the donor host despite any associated fitness costs and provide a transient reservoir of preadapted mutations. ¹

(My emphasis) There’s also theoretical and experimental work that probably addresses how this sort of pressure could drive population-level robustness.  For example, while heterogeneity is linked to fitness in HIV,³ Claus Wilke says:

Virus strains with a history of repeated genetic bottlenecks frequently show a diminished ability to adapt compared to strains that do not have such a history.⁴

I don’t know that work as well as I’d like to, but I think it’s probably relevant when considering local and global evolutionary pressures on the virus.

1. Hoelzer, K., Murcia, P., Baillie, G., Wood, J., Metzger, S., Osterrieder, N., Dubovi, E., Holmes, E., & Parrish, C. (2010). Intrahost Evolutionary Dynamics of Canine Influenza Virus in Naive and Partially Immune Dogs Journal of Virology, 84 (10), 5329-5335 DOI: 10.1128/JVI.02469-09[↩][↩][↩][↩]
2. Kuroda, M., Katano, H., Nakajima, N., Tobiume, M., Ainai, A., Sekizuka, T., Hasegawa, H., Tashiro, M., Sasaki, Y., Arakawa, Y., Hata, S., Watanabe, M., & Sata, T. (2010). Characterization of Quasispecies of Pandemic 2009 Influenza A Virus (A/H1N1/2009) by De Novo Sequencing Using a Next-Generation DNA Sequencer PLoS ONE, 5 (4) DOI: 10.1371/journal.pone.0010256[↩]
3. Bordería AV, Lorenzo-Redondo R, Pernas M, Casado C, Alvaro T, et al. (2010) Initial Fitness Recovery of HIV-1 Is Associated with Quasispecies Heterogeneity and Can Occur without Modifications in the Consensus Sequence. PLoS ONE 5(4): e10319. doi:10.1371/journal.pone.0010319[↩]
4. Novella, I., Presloid, J., Zhou, T., Smith-Tsurkan, S., Ebendick-Corpus, B., Dutta, R., Lust, K., & Wilke, C. (2010). Genomic Evolution of Vesicular Stomatitis Virus Strains with Differences in Adaptability Journal of Virology, 84 (10), 4960-4968 DOI: 10.1128/JVI.00710-09[↩]

Indeed, the amount of HIV diversity within a single infected individual can exceed the variability generated over the course of a global influenza epidemic, the latter of which results in the need for a new vaccine each year. ¹

That was said as part of a discussion on HIV vaccines, but let’s think about it from the influenza side.  Why is it true? Why doesn’t influenza have as many variants as HIV?

(Update: Another paper also looks at this question and points to some interesting explanations; I talk about that paper here.)

We know that influenza, like other RNA viruses, is prone to mutation (that is, it has an error-prone polymerase). Depending how you measure it, it’s likely that almost every new influenza genome has at least one mutation in it, meaning that every new infected animal or person should be be generating thousands upon thousands of new influenza variants.

Globally, of course we do see thousands of new flu variants each year.² But, based on replication fidelity, you’d expect to see a lot more — maybe not quite as many as HIV, but not far from it.

This is also true on a much smaller scale, looking within infected individuals (animals or people).  Even using modern deep-sequencing techniques (like those used in some of the HIV analyses) that should in theory be able to detect large numbers of mutations, there are fewer than you might expect based on the known replication fidelity — far fewer variants than in HIV:

Inasmuch as the mutation rate for type A influenza viruses is estimated at one nucleotide change per 10,000 nucleotide during replication and most infections are caused by as many as 10 to 1000 virions which likely possess varying numbers of nucleotide differences in their genomes, one can expect that each influenza A virion is possibly a quasispecies. However, we identified relatively few quasispecies – probably because the currently available sequence analysis software do not allow robust quasispecies analysis and extensive manual curation is necessary. We believe that with the help of improved bioinformatic tools we would detect more quasispecies populations in our sample sets.  ³

H1N1 (swine-origin influenza virus)

I don’t know enough about the computational side to comment on their bioinformatics point.  Another recent paper⁴ uses a similar approach and (at least at first) seems to reach an more conservative conclusion.  They talk about “quasispecies”, but they seem to be using the term rather loosely, to describe just a handful of distinct genomic sequences. These sequences differ by, for example, a single base (and a single amino acid) in the HA, where one of the sequences was present at about 75% of the sequences, and the other at about 25%. To me that’s not really a “quasispecies” — a quasispecies is something that needs to be defined by an average sequence even though the vast majority of the genomes are different from that average. (Here and here are Vincent Racaniello’s explanations at The Virology Blog.)  Two sequences is just two sequences.

However! The authors do make their data available. I don’t have time to do a detailed look, but from what I think is a very conservative analysis, in one stretch of just 25-40 bases some 5-10% of the genomes have at least one mutation.⁵ If that’s roughly true across the whole genome, then each genome would have, what, maybe a half-dozen mutations on average. That, to me, really is a quasispecies.

(Do note that this is not the mutation frequency for any individual residue. No single point [with the two or three exceptions that the authors focused on] is mutated at much more than one in a thousand, and most probably more like one in many thousand, which is about what you’d expect. )

There are a myriad of complicating factors separating the error frequency in these genomes from the raw error rate of the viral polymerase. A couple of huge ones: These viruses had undergone a bunch of replications in the host – this isn’t the error rate per replication cycle, it’s the cumulative error rate after many cycles. The virus was from a patient who had died with (and probably of) the virus, and though we don’t know how many time the original infecting virus had replicated it was at least a half dozen cycles, perhaps two or three times that.

Influenza virion

On the other hand, during those replication cycles, many mutations (quite likely even the great majority of them) would have been deleterious or outright defective, so most of the mutations would have never propagated but would have just silently disappeared and not been counted at the end.

The most interesting point is that these mutations aren’t arising in a vacuum. Thinking now about which mutations survive and get detected, not the baseline rate of mutation formation: The variants are forming are in an environment that’s designed to be very hostile to viruses. Mutations are going to undergo selection by the immune system.

This is one place where influenza is going to experience a very a different set of pressures than HIV. HIV persists in the presence of the adaptive (T cell and antibody-based) immune response, whereas as the adaptive response kicks in for flu the virus gets evicted. HIV therefore not only have a much longer period (years instead of days) to throw out mutations, it also is shaped by the immune response. By comparison flu would probably only have a couple of replication cycles in the presence of an adaptive response.

Changes in the virus that accumulate over the handful of replication cycles would reflect a strong selection pressure. The vast majority of mutations, even those that aren’t completely defective, are going to be less fit than the original virus and won’t accumulate. Knowing which mutations do accumulate should be very interesting because it may tell us what the virus is going through in the host.  That’s what the authors of this paper focused on — the one particular site that had a much, much higher variant  frequency, more like 25% of the genomes.  The assumption is that this arose during the infection and was positively selected for. ⁶

The variants that replicate best in a host may be quite different from those that are effectively transmitted. That is, there may be multiple sources of selective pressure, of which we have previously mainly only seen transmission pressure (because that’s the main one that will accumulate in a population, because transmission represents a bottleneck in the virus’s evolution [link to The Virology Blog]).  The particular HA variant that was picked up here (that apparently accumulated during the infection) is rare globally. Is that a version of the HA that’s more efficient within a host, but that doesn’t transmit as well?

I think a major reason for the difference between HIV and influenza variant accumulation is the difference between within-host and between-host (transmission) selection.  HIV spends long, long periods within a single host, thousands of replication cycles, accumulating mutations.  The transmission bottlenecks come at much longer intervals and have a much larger accumulated population to work with.  Influenza has a comparatively brief period within the host, only a handful of replications before a new transmission bottleneck hits. ⁷

This sort of deep sequencing experiment on influenza will probably be improved over the next few years, and I’ll be very interested to see just how much variation there really is within on flu-infected host.

1. Walker, B., & Burton, D. (2008). Toward an AIDS Vaccine Science, 320 (5877), 760-764 DOI: 10.1126/science.1152622[↩]
2. More correctly, I suppose, we infer the presence of thousands of new variants based on the hundreds of them that we see, and knowing that we are only examining a tiny fraction of all the flu cases that are out there.[↩]
3. Ramakrishnan, M., Tu, Z., Singh, S., Chockalingam, A., Gramer, M., Wang, P., Goyal, S., Yang, M., Halvorson, D., & Sreevatsan, S. (2009). The Feasibility of Using High Resolution Genome Sequencing of Influenza A Viruses to Detect Mixed Infections and Quasispecies PLoS ONE, 4 (9) DOI: 10.1371/journal.pone.0007105[↩]
4. Kuroda, M., Katano, H., Nakajima, N., Tobiume, M., Ainai, A., Sekizuka, T., Hasegawa, H., Tashiro, M., Sasaki, Y., Arakawa, Y., Hata, S., Watanabe, M., & Sata, T. (2010). Characterization of Quasispecies of Pandemic 2009 Influenza A Virus (A/H1N1/2009) by De Novo Sequencing Using a Next-Generation DNA Sequencer PLoS ONE, 5 (4) DOI: 10.1371/journal.pone.0010256[↩]
5. I extracted the FASTQ data containing the short sequence reads matching influenza sequences from the supplemental PDF, converted it to FASTA, and used xdformat to move it into a BLAST database. Then I grabbed 40 bases from the genbank sequence CY045951.1, the PB2 segment of the closest-match influenza strain, choosing a region (positions 2151-2190) with very high coverage, and BLASTed this sequence against the short sequence data, using parameters such that I retrieved sequences that match at least 25 of 40 positions.  Of the  ~2050 hits I retrieved, about 120 had at least one internal mismatch. I can’t distinguish these from sequencing errors, but I think it’s much higher than you’d expect from sequencing error.  And I hope that my conservative approach (for example, I would have discarded mismatches at the ends of the hits) would balance out that source of confusion. [↩]
6. One point, by the way, that the authors didn’t cover was the possibility that this patient had actually been initially infected with more than one viral sequence.  We do know that a significant number of flu cases are doubly infected. The fact that the minor variant is a very unusual strain makes this less likely, but not impossible.[↩]
7. And I think it’s fair to say that the global population-based HIV variation — the transmission-selected amount of variation, as opposed to the vast within-individual variation — is rather more comparable to that of influenza.[↩]

“Episode de la fièvre jaune”

By analyzing hepatitis C virus genome sequences, you can trace the virus’s history through its spread by the slave trade, and linked 19th-century health models in different countries to viral spread and transmission. Similarly, by looking at leprosy DNA, you can track its spread along the Silk Road and along slave routes.

Yellow Fever was one of the most dreaded plagues of the 18th and 19th centuries, waning only after it was understood to be mosquito-borne, so that mosquito control pushed the virus back. It’s still prevalent in Africa and in some parts of South America, though. Yellow Fever virus, too, originated in Africa and was spread to the New World through the slave trade:

The most commonly cited hypothesis of the origin of YFV in the Americas is that the virus was introduced from Africa, along with A. aegypti,¹ in the bilges of sailing vessels during the slave trade. … We estimate that the currently circulating strains of YFV arose in Africa within the last 1,500 years and emerged in the Americas following the slave trade approximately 300–400 years ago. These viruses then spread westwards across the continent and persist there to this day in the jungles of South America.²

Mosquitoes aren’t merely passive carriers of the Yellow Fever virus. The virus actively infects the mosquitoes as well as their mammalian host, entering the insect gut, replicating and multiplying in various organs until it reaches the saliva, from which it can re-infect mammals³ when the mosquito bites and injects its anticoagulant saliva.

“Latest from the front — our friends the mosquitoes preparing and off for the summer campaign” (Harper’s Weekly, 1873)

Another pattern is possible: The virus could also be spread vertically, from the mosquito to its egg, infecting the newborn mosquito before it hatches. However, although this was shown to happen as long ago as 1905,⁴ just after mosquitoes were proven to be carriers, it hasn’t been very clear if this is a significant part of the natural viral cycle or if it’s more of a lab curiosity:

Although transovarial transmission of YFV has been demonstrated, the relative importance of this in maintaining the transmission cycle is unknown. ⁵

Now, genome sequence analysis suggests that in fact transovarial spread of Yellow Fever virus may well be common and important in the viral life cycle.⁶

This was based on comparisons of Yellow Fever virus genome sequences over time, with those of a close relative, Dengue virus. Dengue and YFV probably arose about the same time, in the same area, and were both spread along the slave trade. But Dengue seems to have diversified much more than YFV:

… it is intriguing that the overall age of YFV (emergence within the last 2,500 years) is broadly similar to the time of origin of the four DEN viruses. Hence, YFV and DENV seem to have radiated at approximately the same time. However, since this time, DENV has differentiated into four antigenically distinct viruses while YFV is still classified as a single serotype.⁶

(This is actually clinically very significant, because the most severe form of Dengue disease is caused by sequential infection with two different Dengue serotypes.) In fact, in general YFV shows a much slower rate of evolution over time than Dengue — about 5-fold slower per year. The authors consider a reject a number of explanations for this — it’s not that they have different mutation rates, because their raw mutation rates are probably quite similar; it’s not that they infect different hosts, because they have very similar insect and mammalian hosts; and so on — and finally suggest that the difference may be because YFV spends a significant part of each year lying more or less dormant in mosquito eggs:

In particular, it is possible that a mechanism of vertical transmission, such as transovarial transmission where the virus may remain quiescent in mosquito eggs for many months, plays a more important role in YFV than in DENV⁶

As a result of this quiescent period, YFV would simply have fewer replication cycles per year than does Dengue, and so it appears to evolve more slowly. For this to be detectable at this level, transovarian transmission would have to be a fairly common event, not just a once-in-a-while half-accidental option.

1. A. aegypti is the mosquito that is most involved in spreading the virus[↩]
2. Bryant, J., Holmes, E., & Barrett, A. (2007). Out of Africa: A Molecular Perspective on the Introduction of Yellow Fever Virus into the Americas PLoS Pathogens, 3 (5) DOI: 10.1371/journal.ppat.0030075[↩]
3. Mainly primates, for functional transmission[↩]
4. Marchous E, Simond PL. 1905. La transmission hereditaire du virus de la fievre jaune chez la Stegomyia fasciata. C. R. Soc. Biol. 59:259[↩]
5. Barrett, A., & Higgs, S. (2007). Yellow Fever: A Disease that Has Yet to be Conquered Annual Review of Entomology, 52 (1), 209-229 DOI: 10.1146/annurev.ento.52.110405.091454[↩]
6. Sall, A., Faye, O., Diallo, M., Firth, C., Kitchen, A., & Holmes, E. (2009). Yellow Fever Virus Exhibits Slower Evolutionary Dynamics than Dengue Virus Journal of Virology, 84 (2), 765-772 DOI: 10.1128/JVI.01738-09[↩][↩][↩]

“Batrachia”, by Ernst Haeckel (Kunstformen der Natur, 1904)

There’s a constant viral assault on us humans, as there is on just about all other species. We as a species have to contend not only with the vast pool of human pathogens, those viruses that constantly circulate among humanity; but also with the continual probes on our defenses from other viruses, viruses that normally infect other species.  All of us are exposed to these on a regular basis: Dog and cat viruses, mouse viruses, crow and pigeon viruses, bat viruses, not to mention the ocean of insect and fungus and amoeba and plant viruses.

Almost all of these assaults don’t even scratch our defenses.  The viruses can’t even enter our bodies, and if they do then they can’t enter our cells, and if they do they can’t replicate in our cells, and if they do then they can’t  …

Most viruses, in other words, can’t effectively jump species.  Even when they do, they’re usually not well adapted to the new species, and they can’t establish a productive chain of infections. Even if they cause a disease, they burn themselves out, infecting fewer and fewer individuals each round of infection, until they disappear.

But every so often, in a tiny minority of cases, the virus does get a foothold.  This is one of the ways that “emerging infections” get started.  It covers things like HIV, SARS, parvovirus of dogs, Ebola, and of course the new H1N1 swine-origin influenza virus (SOIV), among many others.

Why did these guys take off, when so many other viruses failed? Why did SOIV infect people last year, while decades of exposure to pigs and swine H1N1 influenza viruses didn’t lead to earlier pandemics?  Basically, we don’t know, and we’d really, really like to know, so we have a chance of predicting the next SOIV or HIV before it’s a pandemic.

OK, so that explains why I’ve written a fair number of posts here on species-jumping in viruses (here, here, here, here, and here), and partly explains why I want to mention a new paper from Bertram Jacobs‘ lab¹.  (The rest of the reason is, as always, that I just think it’s  cool.)  I’m not sure why Jacobs has done this particular project, because he’s more of an interferon guy, but he’s looked at the origins of ranaviruses and finds evidence for lots of species shifts in their history.

“The Smooth Terrapin (Emys terrapin)”, by James Dekay (Zoology of New York; or, The New York fauna, 1843)

Ranaviruses are probably best known as frog viruses, but they infect a bunch of cold-blooded animals — fish, frogs, salamanders, turtles, and so on — and several of them are causes of emerging infectious disease (as I discussed last time I talked about ranaviruses, here).  Jacobs’ group looked at about a dozen of them whose genomes are completely sequenced², and tried to put together their evolutionary history, which turns out to involve all kinds of cross-species jumps:

…we hypothesize that the most recent common ancestor of the ALRVs was an ancestral fish virus …  Both of these hypotheses suggest that for the majority of evolutionary time vertebrate iridoviruses were confined to fish, and much more recently, there appear to have been at least three species jumps, from fish to frogs, from fish to salamanders, and from frogs to reptiles, and perhaps as many as four species jumps, including a jump from tetrapod amphibians back to fish. It is tempting to speculate that activities associated with human harvesting of aquatic organisms during the past 40,000 years led to the more common recent jumping of ranaviruses among aquatic organisms.¹

(My emphasis) They don’t offer any specific reasons why the ranaviruses should be able to leap from species to species like the chamois of the Alps, but they do make the general point that these viruses tend to be rather promiscuous to start with.  Not only are closely-related viruses able to infect different hosts, but even the same viruses often are able to infect a wide range of species; the fish virus they sequenced in this paper, epizootic hematopoietic necrosis virus, can infect a half-dozen different species of fish.  They raise an interesting comparison:

In addition, the ability of this group of viruses to infect such a wide variety of host species suggests that more host shifts are likely. Therefore, it is important that we understand more of the evolutionary traits of this unique group of viruses, as there is no other closely related group of viruses that infect such a broad group of hosts, with the possible exception of the orthomyxoviruses.¹

Orthomyxoviruses, of course, include influenza viruses, which notoriously infect humans, pigs, ducks, chickens, wild waterfowl, horses, and dogs; and you’ll recall all the reports during the epidemic phase of SOIV of the virus infecting all kinds of other pets and domestic animals.  Influenza viruses are apparently evolving at an even faster pace than the ranaviruses, and experimenting with even more species; but there may be lessons for us (as influenza hosts) in the ranaviruses.

1. Jancovich, J., Bremont, M., Touchman, J., & Jacobs, B. (2009). Evidence for Multiple Recent Host Species Shifts among the Ranaviruses (Family Iridoviridae) Journal of Virology, 84 (6), 2636-2647 DOI: 10.1128/JVI.01991-09[↩][↩][↩]
2. Including epizootic hematopoietic necrosis virus, whose genome they sequenced themselves[↩]

Man chasing rabbit (From “Fliegende Blätter”, Munich, 1889)

Everyone knows about rabbits in Australia. Introduced in the mid-1800s, they multiplied ridiculously and ate their way across the country, leaving barren devastation behind them.

Myxomavirus, a poxvirus that originated in South America, was introduced in the early 1950s and temporarily controlled the rabbit population, cutting their numbers by 85% (to a mere hundred million rabbits); but the rabbits evolved some resistance, and the virus evolved somewhat reduced virulence, and after about 15 years the rabbit population started to build up again. (I’ve talked about myxomatosis and rabbit control here and here.)

Myxomavirus isn’t a natural pathogen of European rabbits; its natural hosts are American rabbits, in which it causes a much more mild disease. It’s a virus that jumped into a new species, was very virulent in that new species, and then became less so over 15 years or so of transmission in the new species.

Myxomavirus is often used as an example of a virus that evolves toward avirulence, with the message usually being that this is the usual path of evolution. For example, you’ll see comments like, “Typically, viruses that rapidly kill their host have a very short history, as they rapidly run out of places to reproduce.” As I’ve tried to point out several times (see the myxomavirus links above), this isn’t true; pathogens in general evolve toward improved transmission, not reduced virulence. In many cases, reducing virulence does enhance transmission, but it’s not the only path. And myxomavirus doesn’t even support the claim all that well, given that even the “low-virulence” strain that’s out there now still has a mortality rate about the same as Ebolavirus, or smallpox.

OK, hold that thought.

Extermination of rabbits in California (From “The Picture Magazine,” 1894)

Myxomavirus worked well to control rabbits for a while, then became less effective. In 1995, a new virus was introduced into Australia and New Zealand,¹ a calicivirus that causes Rabbit Hemorrhagic Disease, called (with stunning originality) Rabbit Hemorrhagic Disease Virus (RHDV). Where did RHDV come from?

Basically RHDV is the opposite of myxomavirus. The parent of RHDV is a natural virus of European rabbits, but it causes little or no disease. RHDV is a natural mutation of this virus, and it has very high virulence – the opposite of the viruses-evolve-to-low-virulence claim. Even with the help of Australian farmers, RHDV is highly successful. It spread around the world in the mid-1980s after first appearing in Chinese rabbits in 1984.

One of the most intriguing aspects of RHDV evolution is that this virus appears to have maintained its very high virulence during the 25 years since it emerged. At face value this suggests that virulence is adaptive for transmission. ²

Rabbit’s eye (Max Brödel, 1932)

In fact, it’s been believed that the virulent RHDV is such a successful mutation that it arose several times, independently, from the mild parent virus. (Compare to the feline infectious peritonitis story, where the prevalent explanation for the appearance of a virulent FIP infection is that innocuous gastrointestinal coronaviruses, that are widespread in cats, mutates to form the virulent form; and this mutation is independent for each cat, rather than arising once and then spreading.)

At least, that’s been the established explanation, but a recent paper² shows that a couple aspects of that aren’t correct. The authors looked at genome sequences of many rabbit caliciviruses (the mild parent calicivirus as well as the virulent RHDV) to track its origins and spread. RHDV is indeed a recent mutation from a mild parent virus; that much is correct. But RHDV probably only arose once, not multiple times, and the origin of RHDV was well before 1984) when it was identified as a disease.

The independent-mutation hypothesis was based mainly on finding RHDV-related viruses circulating in Europe in the 1950s:

Prior phylogenetic work led to suggestions that RHDV with sequences closely related to those that emerged from China in 1984 were circulating harmlessly in the United Kingdom and other European localities during the 1950s; hence, it was suggested that virulence emerged at least twice during the late 20th century: once in Europe and once in China. ²

But Kerr et al looked more closely at these early isolates, and don’t think they’re real:

… we show here that the sequences from the 1950s and 1970s from the United Kingdom appear to be modern contaminants: given the rate of RHDV evolution documented here and that of RNA viruses more generally, these early RHDV sequences are expected to be far more divergent from their modern counterparts. ²

(Compare to the influenza database, where there also seems to be a significant level of mis-identified virus.)

So RHDV probably only originated once, which is a little more reassuring than the notion that this high virulence is so easy to achieve that it can appear many times over a short period. Did the original mutation appear around 1984, when the disease was noted? The authors identified 4 distinct strains of RHDV and noted:

A common feature of all of these groups is that many lineages likely originated during the 1970s, suggesting that there was a period of viral radiation at this time…. Crucially, this also means that there were already multiple separate lineages of RHDV before the documented emergence of RHD in China in 1984.  … This implies either that high virulence evolved multiple times in multiple viral lineages close to 1984 or (more plausibly) that virulence emerged earlier in the 20th century but the disease was not documented until 1984 when the trade in rabbits provided the opportunity for RHDV to spread from an established, but apparently cryptic, transmission cycle. … Therefore, we propose that the most likely scenario is that virulent RHDV strains evolved once, early in the 20th century, but were not detected until 1984. ²

(My emphasis) This seems, at first glance, surprising. RHDV kills almost all of the rabbits it infects. Wouldn’t you notice it if all your rabbits suddenly fell over dead? How could RHDV circulate for years or decades without being detected? Kerr et al make some points about the nature of the disease (it can infect very young kits without killing them, for example), but also comment:

Given the difficult sociopolitical conditions in China and neighboring countries in the first half of the 20th century, it is plausible that a virulent disease in rabbits was able to evolve in this region without leaving a clear record. ²

The pandemic swine-origin H1N1 probably has been circulating in swine for quite a while (years? decades?) without being picked up, and probably circulated in humans in Mexico for months before it was detected there. It’s pretty easy to believe that rabbits in China during the Cultural Revolution didn’t get as much attention as pigs in the US in the 2000s.

So, an interesting story in its own right, especially thinking about evolution of virulence in pathogens; and also, a story that probably reflects some important lessons for human health.

1. It wasn’t supposed to be introduced then; it was penciled in for few years later, after more study, but somehow it jumped from the island where it was being studied to the mainland. The usual explanation is “via insects”, but of course one has to wonder if some Australian farmers didn’t help the insects along some. As I recall from the news reports at the time, the “accidentally introduced” virus spread throughout Australia very fast, almost as if dead rabbits were being carted around by car or something.[↩]
2. Kerr, P., Kitchen, A., & Holmes, E. (2009). Origin and Phylodynamics of Rabbit Hemorrhagic Disease Virus Journal of Virology, 83 (23), 12129-12138 DOI: 10.1128/JVI.01523-09 [↩][↩][↩][↩][↩][↩]

Leprosy is a fascinating disease for many reasons.  Historical, because, well, it’s leprosy.  Genetic, because the bacterium is apparently derived from a single clone that infected humans some 4000 years ago,¹ and that has undergone “massive gene decay” in the process of becoming an obligate pathogen:

Thus, since diverging from the last common mycobacterial ancestor, the leprosy bacillus may have lost more than 2,000 genes. ²

Immunological, because as mycobacteria, leprosy and tuberculosis may have an entire branch of the immune system dedicated to their control and destruction.  Epidemiological, because leprosy is one of the very few diseases that has the potential for elimination without vaccines.  And now let’s add phylogeography and anthropology to the list, with a paper that offers a detailed analysis of leprosy’s migration through humanity. ³

This was done by genetic analysis, tracking through sub-types of leprosy in various areas, both modern and ancient — the latter being “obtained from leprosy graveyards in Croatia, Denmark, Egypt, England, Hungary and Turkey“, and allowing the authors to determine the strains of leprosy that circulated as much as 1500 years ago.  Their conclusions (building on and extending earlier work):

• The progenitor of leprosy arose in East Africa
• New strains then spread into Asia, through two different routes: One northern route, and one southern
• The Southern route into Asia was probably the Silk Road: “the trade route between Europe and Asia known as the Silk Road appears likely to have been a means of transport and disease transmission“.  They point out that this is the opposite path of the Black Plague, which likely spread from Asia to Europe along the Silk Road.
• Another strain of leprosy moved from East Africa westward into the Middle East and Europe
• This strain in turn spawned strains that are found in West Africa and countries linked to West Africa by the slave trade.  (Compare to the phylogeography of hepatitis C, among other diseases spread by slavery)
• Leprosy in North America came from relatively recent European immigrants, rather than coming along with the original Bering Strait peoples.

One interesting conclusion is that the genome decay of M. leprae is much older than humans (occurring over a million years ago, whereas humans are only a few hundred thousand years old), even though the genetic evidence says the present bacteria were clonal just a few thousand years ago.  They suggest that

Alternatively, the genome decay could well be ancient, but M. leprae may only recently have become a human pathogen. For instance, it is conceivable that an ancestral form of M. leprae infected an invertebrate host such as an insect, which later acted as a vector for transmitting the bacillus to humans.  ³

1. Monot, M. (2005). On the Origin of Leprosy Science, 308 (5724), 1040-1042 DOI: 10.1126/science/1109759[↩]
2. Cole, S., Eiglmeier, K., Parkhill, J., James, K., Thomson, N., Wheeler, P., Honoré, N., Garnier, T., Churcher, C., Harris, D., Mungall, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Duthoy, S., Feltwell, T., Fraser, A., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Lacroix, C., Maclean, J., Moule, S., Murphy, L., Oliver, K., Quail, M., Rajandream, M., Rutherford, K., Rutter, S., Seeger, K., Simon, S., Simmonds, M., Skelton, J., Squares, R., Squares, S., Stevens, K., Taylor, K., Whitehead, S., Woodward, J., & Barrell, B. (2001). Massive gene decay in the leprosy bacillus Nature, 409 (6823), 1007-1011 DOI: 10.1038/35059006[↩]
3. Monot, M., Honoré, N., Garnier, T., Zidane, N., Sherafi, D., Paniz-Mondolfi, A., Matsuoka, M., Taylor, G., Donoghue, H., Bouwman, A., Mays, S., Watson, C., Lockwood, D., Khamispour, A., Dowlati, Y., Jianping, S., Rea, T., Vera-Cabrera, L., Stefani, M., Banu, S., Macdonald, M., Sapkota, B., Spencer, J., Thomas, J., Harshman, K., Singh, P., Busso, P., Gattiker, A., Rougemont, J., Brennan, P., & Cole, S. (2009). Comparative genomic and phylogeographic analysis of Mycobacterium leprae Nature Genetics, 41 (12), 1282-1289 DOI: 10.1038/ng.477 [↩][↩][↩]

There’s been recent excitement over the discovery of bornaviruses fixed in the human genome^{1, 2}.  Exciting and unexpected as that is, as usual, the insects are way ahead of us.  The genome of a parasitoid wasp has poxvirus sequences in it!

Detecting ancient lateral transfers is more problematic. By examining protein domain arrangements in Nasonia relative to other organisms, we uncovered an ancient lateral gene transfer involving Pox viruses, Wolbachia, and Nasonia. Thirteen ANK repeat–bearing proteins encoded in the N. vitripennis genome also contain C-terminal PRANC (Pox proteins repeats of ankyrin–C terminal) domains. This domain was previously only described in Pox viruses, where it is associated with ANK repeats and inhibits the nuclear factor ΚB (NF- ΚB) pathway in mammalian hosts …³

These parasitic wasps are not the same family as the magnificent braconid parasitic wasps that have developed a symbiotic relationship with polydnaviruses (see my posts here and here), and braconids’ incorporation of nudivirus genomes already trumps the bornavirus findings.  But still.  Poxviruses!

I don’t think we have any functional information on what the Nasiona are doing with the poxvirus genes here, and I know very little about wasp biology, but given that:

• in mammals these genes are  inhibitors of the innate immune response,
• the innate immune response is relatively  conserved from insects to humans, and
• Braconid wasps use their symbiotic viruses to inhibit their prey’s immune responses,

I wonder if the Nasonia have independently come up with the same idea as Braconids, and incorporated a viral immune evasion molecule to use in their venom to suppress their prey’s immune response to the wasp’s eggs and larvae.

1. Original paper in Nature[↩]
2. See commentary in the New York Times;  the Virology Blog; and Not Exactly Rocket Science[↩]
3. The Nasonia Genome Working Group (2010). Functional and Evolutionary Insights from the Genomes of Three Parasitoid Nasonia Species Science, 327 (5963), 343-348 DOI: 10.1126/science.1178028[↩]

Geographic distribution of H5N1 highly pathogenic avian influenza viruses (HPAIVs) used in this study. Darkened provinces indicate locations of virus isolation.

“We found several patterns that suggest one general model of evolution in this viral system: 1) within regions, viral mixing in poultry moves toward heterogeneity and the emergence of local types; 2) differentiation was centered around regional viral hubs located at centers of human and bird population density; and 3) evolution occurs because of relative isolation of the hubs, most likely fed by the abundant supply of domesticated poultry (and people) at the hubs. The analysis thus suggests that at the scale of neighboring city hubs and the intervening hinterland, evolution of H5N1 follows the pattern described by classical theory of genetic differentiation due to isolation by distance.” ¹

Genetic versus geographic distance of HK821-like HPAIVs in Vietnam.

This is in Vietnam, and the basic finding was that H5N1 viruses isolated in Vietnam show signs of local evolution, in that the viruses cluster into local sub-strains in different areas of the country.  I’m not all that knowledgeable about H5N1 spread, but I had thought that infection of wild, especially migratory, birds would be an important factor in spreading H5N1 between chickens.  If I’m interpreting this paper right, it looks as if H5N1 is mostly circulating within local regions, within the chicken population, and distant spread isn’t a major factor. That has obvious implications for control of the virus.

1. Carrel, M., Emch, M., Jobe, R., Moody, A., & Wan, X. (2010). Spatiotemporal Structure of Molecular Evolution of H5N1 Highly Pathogenic Avian Influenza Viruses in Vietnam PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008631[↩]

As if understanding this complex evolutionary puzzle were not already sufficiently challenging, we have learned recently that two types of adaptive immune system have evolved in vertebrates: a recently recognized system in jawless vertebrates (hagfish and lamprey) and the more familiar adaptive immune system of jawed vertebrates. … This leads to the conjecture that two interactive lymphocyte arms are a fundamental feature of the adaptive immune system that was selected to provide balance and self-regulation.

Cooper, M., & Herrin, B. (2010). How did our complex immune system evolve? Nature Reviews Immunology, 10 (1), 2-3 DOI: 10.1038/nri2686

HA structure showing mutating amino acids1

Anyone who’s taken a virology class, and many who haven’t, know about “antigenic drift” and “antigenic shift”. These are usually used to explain influenza virus changes over time (although of course the same concepts apply to many other viruses). Antigenic shift refers to large, abrupt changes in the virus;² antigenic drift refers to smaller changes. Antigenic shifts are associated with pandemic influenza, as the pre-existing immune responses to influenza from previous years aren’t protective any more (because the virus has shifted away from them). Antigenic drift doesn’t let the virus escape immune control altogether, but does give the virus an advantage in infecting people — presumably, people with strong responses are still protected, but those whose exposure was maybe a few years ago, or who happened to make a weaker antibody response, to the previous virus, would be susceptible to the new virus but protected against the original. Antigenic drift happens all the time, and new drifted variants of influenza take over every year or two, which is why we need new seasonal flu vaccines on a regular basis.

What drives antigenic drift? The simple answer is that it’s driven purely by immunity. According to this notion, viruses that are resistant to being neutralized by antibody are most able to replicate and transmit to new hosts. Jon Yewdell’s group, however, has just revisited antigenic drift,¹ and propose a somewhat different model: Antigenic drift is the result of viruses cycling between immune and non-immune hosts, and it’s almost a side effect of the way the virus interacts with cells.  (Unusually for Jon, I don’t htink he coined any new and exciting acronyms for his new model.)

Model for antigenic drift selection (click for larger version)1

Normally, influenza virus doesn’t “want” to bind very strongly to its cellular target, ³ because then it’s harder for newly-formed viruses to escape from the cell (because it binds to the receptors on the way out, as well). But in the presence of neutralizing antibody, the virus needs to bind more strongly to the receptor to overcome the effects of antibody binding. Yewdell’s group argue that it’s this whip-saw effect that pushes long-term changes in antigenicity:

Thus, antigenic drift can be a by-product of Darwinian selection for mutations that optimize host cell receptor binding during influenza A virus transmission between immune (increased receptor binding) and naïve individuals (decreased receptor binding). ¹

One difference between Jon’s model and the standard concept is that with the latter, you’d expect that there would be more antigenic drift as immunity increases among the population. Yewdell’s model, though, predicts that sequential passage through immune and non-immune individuals drives antigenic drift. This actually leads to an important prediction:

In our model, antigenic drift is accelerated by sequential passage of influenza A virus between immune and nonimmune individuals, which in the human population are nearly all children. Therefore, decreasing the naïve population size by increasing pediatric influenza A virus vaccination rates will likely slow antigenic drift and temporally extend the effectiveness of influenza vaccines.¹

(My emphasis.)  They also point out that, because changes in antigenicity run in parallel with changes in receptor binding affinity, antigenic drift can itself could be pushing other changes in virus personality. That’s because the drift changes push changes in receptor binding, which in turn alters the cells to which the virus can interact; and changing the cells that the virus infects will inevitably change the nature of an infection as well. For example, does the virus best infect cells of the upper respiratory tract — leading to coughs and sniffles — or the lower respiratory tract — leading to pneumonia.

1. Hensley, S., Das, S., Bailey, A., Schmidt, L., Hickman, H., Jayaraman, A., Viswanathan, K., Raman, R., Sasisekharan, R., Bennink, J., & Yewdell, J. (2009). Hemagglutinin Receptor Binding Avidity Drives Influenza A Virus Antigenic Drift Science, 326 (5953), 734-736 DOI: 10.1126/science.1178258[↩][↩][↩][↩][↩]
2. “Changes” here mean changes in the ways the immune system responds to the virus — hence, “antigenic” changes. In practice, the changes are antibody-based changes, although in principle antigenic shift and drift could also refer to T cell-based recognition.[↩]
3. The influenza hemagglutinin, HA, protein binds to sialic acid on cells of the respiratory system.[↩]