There are two aspects about virology that constantly amaze me: How much we know about viruses, and how little we know about viruses.

Adenovirus research offers examples of both. Adenoviruses are probably among the best-studied virus groups.¹ We really do know an amazing amount about them. But it was only last year that Linda Gooding’s group offered the most convincing demonstration yet that adenoviruses actually establish a truly latent infection — a really basic aspect of their lifestyle, ² and a new paper from her group³ is looking at some equally-basic implications of that finding. (I talked about Gooding’s earlier latency finding here.)

It’s been known pretty much since day 1 that adenoviruses persistently infect tonsils;⁴ that was why they were first isolated, when the virus grew out of apparently-normal tonsil tissue in culture. The critical distinction is between mere “persistence” and true “latency”. In a latent infection, the virus shuts down production of new viruses, and is maintained basically as DNA within the host cell. Persistence is cruder — the virus continues to replicate, but at a low level that balances its destruction. Simplistically, latency is a destruction-free process, while persistence can include viral and cellular destruction.

Adenoviruses establish their latency in tonsils, which of course have lots of lymphocytes, but we usually think of adenoviruses as infecting epithelial-type cells, or hepatocytes, or whatever. Clinically, these guys typically cause cold-type symptoms, which you tend to get from fairly superficial infections of the respiratory tract lining. We don’t tend to think of adenoviruses as effective infectors of lymphocytes, but it turned out that their latent infection was, in fact, in T lymphocytes.  It looks like adenoviruses have one cell type (epithelial-type cells) for a lytic infection that leads to shedding of infectious virus, and another cell type for latent infection, allowing the virus to remain in the host and potentially re-infect an epithelial type later on.

Accordingly, Gooding and her team set up infections of cultured T lymphocytes in vitro, to see what would happen. In particular, they wanted to know whether, and how, the viral replication cycle would be controlled; and whether and how the host cell would be affected by the infection. I will skip over most of their findings and and highlight a couple that surprised me:

(1) The “Occupied!” sign. To get into a cell, adenoviruses usually need to bind to their cellular receptor, the CAR receptor.⁵ But latently-infected cells almost permanently shut off this receptor. For hundreds of days after the initial infection, cells express little or no CAR. The latent virus doesn’t want any competition; it has found a congenial long-term environment, and it doesn’t want some interloper infecting its cozy cell and perhaps destroying it.

There seem to be several mechanisms for the shutdown, but at least part of it is that the virus apparently permanently modifies the host DNA:

CAR synthesis and expression remained repressed even after the viral genome was lost (Fig. 8 and data not shown), suggesting a virus-induced epigenetic change to the cells that does not require the continued presence of the virus.³

And in fact the CAR isn’t the only thing to be modified for this purpose:

Even when CAR levels were restored by transduction with a CAR-containing retrovirus, the previously infected cells could not be reinfected³

We don’t know how the latent viruses were blocking superinfection, but it’s clear that the latent viruses really don’t want company.

(2) Rearranging the furniture.  The latent virus doesn’t stop at hanging an “occupied” sign; it modifies its host cell in other ways as well, apparently again by long-term or even permanent epigenetic modification of the DNA. That means that even after the virus itself is altogether gone, not even latently present, there are modified cells hanging about:

Remembering that adenoviruses infect just about everyone, that may mean that we’re all walking around carrying cells that are tagged and functionally altered by these viruses.

There’s been speculation for many years that adenovirus infection may underlie some forms of human tumors. One argument against this has been that there’s no evidence of adenovirus DNA in tumors, for the most part.⁶ (One rule of thumb in determining if a virus is actually causing a tumor is if it’s actually present in the tumor.) But of course, if adenoviruses leave a permanent scar on cellular DNA that lasts longer than the virus itself, this may not be relevant:

One compelling reason to gain an understanding of this nonlytic infection is the likelihood that adenovirus gene products cause damage to the host cell genome. … While these functions are irrelevant to the lytic infection of epithelial cells where all infected cells die, they are of serious concern when infected lymphocytes have carried the viral genome and survived. … Despite this normal appearance, the cells display altered gene expression long after the virus is lost.³

1. There are over 40,000 papers on adenoviruses, or at least mentioning them, in PubMed.
2. To be fair, it’s been suspected for decades that they do go latent, but that was the first time it was actually proven.
3. Zhang, Y., Huang, W., Ornelles, D., & Gooding, L. (2010). Modeling Adenovirus Latency in Human Lymphocyte Cell Lines Journal of Virology, 84 (17), 8799-8810 DOI: 10.1128/JVI.00562-10
4. I’m going to limit this discussion to the Group C adenoviruses — the latency concept may be true for other groups of adenoviruses but that hasn’t been directly shown.
5. “CAR” stands for the “Coxsackie B virus and Adenovirus Receptor”. Can anyone guess what other virus uses this receptor? Bueller? Anyone?
6. Also, the epidemiological links between tumors and adenoviruses are not very strong, at least in humans.

Normally I don’t talk about research that’s well covered elsewhere, but I like this one so much (and it links back to so many of my earlier posts; check the footnotes for those links) that I’ll make an exception here.  I’d seen bits and pieces of this story, but I didn’t have the big picture until I listened to Carl Zimmer’s¹ latest Meet The Scientist podcast² where he interviewed Nancy Moran.³

So this is kind of about insect immunity. Insects have lots of innate immune responses, the short-term sorts of things that in vertebrates we call ‘inflammation’, but they don’t have the long-term adaptive responses ⁴ that incorporate antibodies and T cells — those systems arose in sharks and their progeny (and, apparently mostly independently, in lampreys and hagfish,⁵ but that’s a different story).

The hallmark of adaptive immunity, in contrast to innate immunity, is its flexibility: Different responses for different agents, and capable of changing as the target changes.  So insects can’t do that, although of course their immune system has worked pretty well for a few hundred million years.

Except aphids have a sort of changable immune response.  How does that work?

Parasitic wasp laying eggs in an aphid (from University of Wisconsin)

This is an immune response, not to bacteria or viruses, but to parasitic wasps.  Aphids are popular targets for some of these wasps: The wasps lay eggs in the aphid, the eggs hatch into baby wasps, and the baby wasps eat the aphids from the inside out until they kill the aphid and then they fly away to predate some more.  Except in some aphids, the baby wasps are killed as they hatch, and the aphids survives to make more aphids.

And this immunity to the wasps is — on a population basis, not an individual basis — rather flexible. Insects in general are good at evolving toxin resistance over years or decades, but aphids have apparently been doing this over millions of years. It turns out that different aphids kill the baby wasps in different ways, using different toxins to do so, and the toxins change over time as well.  So the wasps can’t develop resistance to the toxins.  It’s a little bit — a very little bit — like an adaptive immune system, at least in broad terms.

Not all aphids are immune at all (or there would be no wasps).  You can take susceptible aphids and make them resistant, though. You just have to infect them with a particular bacterium.  This is a symbiotic⁶ bacterium, that only lives in aphids — it’s dependent on the aphid host to provide it with essential nutrients — and these bacteria carry toxin genes. They help their host survive by providing toxin genes, that kill the wasps that parasitize the hosts the bacteria are symbiotic with.

But not so fast! The bacteria don’t naturally have toxins! The toxins come from parasites of the bacteria! There are bacteriophages, viruses that infect the bacteria, that carry the toxins.  When the viruses parasitize the bacteria that are parasitizing the aphids, then the parasitic wasps can’t parasitize the aphids that are hosting the bacteria that are hosting the viruses!

And if you look at the bacteriophages as a population, they have a section of their genome that is highly diverse. That part is the region that carries the toxin. Different phages, different toxins, that can spread to new bacteria and then to new aphids, so the aphids can have a supply of new toxins to take care of newly-resistant wasps.

Just to make this even more complex, you know how the wasps subdue their prey? They inject in a complex mix of toxins that shut down the insect immune system. Guess where those toxins come from?⁷ From the symbiotic viruses⁸  that the wasps have incorporated into their own genomes millions of years ago, that carry immune evasion genes that the wasps have adapted to use to subdue the aphids that carry the bacteria that carry the viruses that provide the toxins that protect the aphids against the wasps that carry their own viruses to attack the aphids.

1. Ed Yong also covered this story last year.
2. By the way, you all should be listening to Meet the Scientist.  Zimmer is not only an excellent writer, he does a really good interview, and the scientists he interviews are all highly articulate and interesting.  Scientists as a group tend to be pretty articulate about their work, because communication is actually part of the job description, but Zimmer is very good about asking the right questions and then getting out of the way.
3. A few of the papers by Moran and her colleagues:
   • Oliver KM, Degnan PH, Hunter MS, & Moran NA (2009). Bacteriophages encode factors required for protection in a symbiotic mutualism. Science (New York, N.Y.), 325 (5943), 992-4 PMID: 19696350
   • Degnan PH, Yu Y, Sisneros N, Wing RA, & Moran NA (2009). Hamiltonella defensa, genome evolution of protective bacterial endosymbiont from pathogenic ancestors. Proceedings of the National Academy of Sciences of the United States of America, 106 (22), 9063-8 PMID: 19451630
   • Degnan PH, & Moran NA (2008). Evolutionary genetics of a defensive facultative symbiont of insects: exchange of toxin-encoding bacteriophage. Molecular ecology, 17 (3), 916-29 PMID: 18179430
   • Degnan PH, & Moran NA (2008). Diverse phage-encoded toxins in a protective insect endosymbiont. Applied and environmental microbiology, 74 (21), 6782-91 PMID: 18791000
4. Earlier posts on insect immunity:
   Invertebrate memory, or wishful thinking?
   “Social immunity” in ants?
   “Social immunity” followup
5. Posts on lamprey and hagfish immunity:
   Same trip, different routes: Lamprey immunity
   Lampreys got antibodies
   Lamprey VLR and antigen binding
   Lamprey immunity, again
6. Posts on other arthropods and their symbiotic viruses and bacteria:
   How the aphid got its wings
   More symbionts and flight
7. To be honest, I didn’t check the kinds of wasp here, so I don’t know for sure that these are among the families of wasps that do this
8. Posts on wasps and their symbiotic viruses:
   Bioweaponized wasps
   Not merely bioweaponized, but mutualistic bioweaponized wasps

PRRS is “porcine reproductive and respiratory syndrome”, which pretty much sums up the disease. It’s caused by — you’ll never guess — Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that emerged in 1987. That was the year I left large animal veterinary practice, so I never had a chance to deal with PRRS clinically.

Twenty-three years may not seem like all that long a time, but if you’re an RNA virus that’s a lot of generation times and a whole lot of time for mutations and evolution, and PRRS viruses are an evolutionarily mess. ¹ There are North American type PRRSV viruses and European type viruses, there are mysterious clusters of related viruses, there are clusters of related diseases, there are thousands of sequences, and it’s just kind of baffling what’s gone on with the whole schtick.

A new paper² has tried to sort out part of the mess by analyzing some 8624 North American-type PRRSV sequences, from nearly a dozen countries, and working out evolutionary relationships between them all. ³ (The focus on the North American series — the Type II PRRSV — is because this group seems to be a more common source of disease; although the European strains are far from rare themselves.)

There were a couple of interesting points that parallel some other viruses:

1. Feral vaccines. It’s already known, or at least strongly suspected, that some of the modified-live PRRSV vaccines have started to go feral on a small scale (not nearly as dramatically as the vaccinia virus I mentioned a while ago), and that’s supported by this genetic analysis:

In the vaccine-associated sublineage phylogenies (data not shown), there were a number of well-supported small clusters that might reflect the small-scale transmission of the vaccine viruses in the field … ²

As well as vaccinia, there are other live vaccines that are known to spread into the population. The sort of limited transmission that seems to be showing up here is more typical of this sort of thing than are the vaccinia instances I talked about before.

2. The amazing flying pigs. Even though this is just one of the two major sub-groups of PRRSV and it’s less than 25 years since it emerged, they came up with nine fairly distinct lineages of the virus (see the figure to the right). As you’d expect the lineages speak to the history of the virus — which is to a large extent the history of the pigs that carried the virus.⁴

This version of the virus probably started out in North America (though how it got there … ?) and then got introduced into other countries on several independent occasions. Two of these introductions were in the late 1980s, shortly after the North American emergence. Aside from that there’s evidence of a bunch of smaller introductions:

… lineage 1 had several Thai sequences clustered with early Canadian sequences … ; lineage 8 contained highly pathogenic Chinese strains and their relatives … ; and lineages 8 and 9 had several Italian isolates which were distributed separately along the phylogeny …, indicating independent introductions of PRRSV from the United States to Italy. ²

They were even able to identify smaller-scale travel patterns, between individual states in the USA:

Iowa plays a central role because its viruses were introduced recurrently to all nine other states (Fig. 5B). The remaining states were not just receiving sites. Their local strains also were transmitted to other states repeatedly, but within a narrower range. … Our phylogeographic analyses reveal, for the first time, an interstate PRRSV traffic network in the United States. … The result also indicates that long-distance spread is a frequent process for PRRSV … ²

This is a reminiscent of the history of the pandemic H1N1 influenza virus, when it was still in swine. (Remember that pandemic H1N1 is genetically  a mixture of a North American swine influenza strain and a Eurasian strain.)  There’s a large national and global traffic in pigs, and even though most countries are reasonably careful in the way they handle incoming pigs it’s not a guarantee against virus introduction. I’m not singling out pigs, either — other kinds of livestock also are global travellers, and obviously so are humans.  But it’s a reminder that it isn’t just humans and their viruses that can quickly travel and spread around the globe.

1. More correctly, our understanding of their evolution is a mess. The viruses are doing just fine.
2. Shi, M., Lam, T., Hon, C., Murtaugh, M., Davies, P., Hui, R., Li, J., Wong, L., Yip, C., Jiang, J., & Leung, F. (2010). Phylogeny-Based Evolutionary, Demographical, and Geographical Dissection of North American Type 2 Porcine Reproductive and Respiratory Syndrome Viruses Journal of Virology, 84 (17), 8700-8711 DOI: 10.1128/JVI.02551-09
3. That’s a lot of viruses, but the sampling is heavily biased to a limited number of places, especially the USA [and especially a few regions within the USA] so it’s probably an underestimate, and maybe a severe underestimate, of the global diversity.

   I didn’t know, by the way, that there’s a PRRSV Database:http://prrsvdb.org/

4. Or of the pig’s fluids. I think that especially in the early days of the emergence, the virus was spread by the boar semen used for artificial insemination.

I’ve also pointed out, though, that actually measuring quasispecies is technically difficult, and measuring it for the larger DNA viruses would be even harder. You’d need to run sequences on many viral genomes, to see how much variation develops over time; and it’s only recently that sequencing tech has approached the point where it’s even thinkable, let alone affordable, to do that:

While large DNA viruses are thought to have low mutation rates, only a small fraction of their genomes have been analyzed at the single-nucleotide level.¹

So maybe DNA viruses might actually form quasispecies, and we don’t know it?

In fact, even for DNA viruses, mutants appear fairly quickly, given the appropriate selection pressure. In principle, these might not even be new mutations, but simply expansion of a particular part of a quasispecies that was already pre-existing. (It’s probably a fairly obvious point, but it’s important to remember that the introduction of new mutants, and their selection and expansion, are completely different processes. Some viruses throw out incredible numbers of mutants, but almost all of them are dead ends that are actively selected against, or at the least not selected for. Other viruses may make far fewer mutants, but given strong enough selection pressure some of these might rapidly take over the population. It’s very tricky to use observed mutations as a measure of mutation frequency, because observation often depends on selection to build up the numbers of the mutant before you can see it.)

At any rate, it’s a fair enough question,² but recently there has been some evidence that supports the concept of DNA virus genome stability. Wayne Yokoyama’s lab has actually sequenced multiple genomes of mouse cytomegalovirus (a large DNA virus — a member of the herpesvirus family) to look at quasispecies.¹

One of the things they did find was a significant number of variations in their stock, compared to the stock they had got it from years before:

… our laboratory’s Smith strain MCMV differed from the previously published Smith strain … There were 452 differences, including 50 insertion/deletions (indels) and 402 single-bp substitutions. … this high number of differences suggested that MCMV mutated in vivo, as we had previously maintained our MCMV stock by in vivo passages. ¹

In other words, the standard methods of maintaining a virus — repeatedly growing new stocks in cells, and using those new stocks to make yet more — allow the accumulation of variations in the genome — which is already well known, of course, but often neglected in lab experiments.  Again, as I point out above, the number of observed mutations we see here doesn’t tell us much about the actual mutation frequency.

How often do mutations arise? By running the virus through cells repeatedly (in vitro, that is) and then seeing how individual clones differed, they determined that there are very, very few mutations per replication. What’s more, and even more impressively, very few mutations appeared after passages through mice (in vivo):

… the remaining 9 mutations allowed us to estimate the mutation rate of MCMV as 1.0 x 10^–7 mutations per bp per day after in vivo passage, very similar to the mutation rate calculated for in vitro passage.¹

(My emphasis) For comparison, the MCMV genome is not quite 250,000 bp long, so we’re looking at around one mutation per 40-50 genomes per day (if I’m dividing right). That’s hundreds of times more stable than most RNA viruses (see the table here³ for some RNA virus error rates).  Still, there’s plenty of room for natural selection in there, because of course there are hundreds or thousands of new MCMV genomes being made per day even in the most conservative estimate (and maybe more like millions or hundreds of millions), so dozens to thousands of them are mutants.

So, not surprisingly, Yokoyama’s group was able to detect a cluster of mutations that were almost certainly selected in the mouse; without going into detail, these mutations were in a viral gene (m157) that’s known to be recognized by the (laboratory) mouse immune system, so it wasn’t surprising that mutants were selected. And such mutants did not appear in mice without the appropriate immune component, demonstrating the role of natural selection in this cluster.

They offer a number of cautions, including one that’s raised in almost all such studies:

One caveat to our mutation analysis is that lethal mutations were probably underrepresented in the final DNA pool since, by definition, they did not propagate. Nonetheless, this limitation is intrinsic to all mutation analysis.¹

Still, the results are solid and reassuring, supporting a basic concept in viral evolution.

1. Cheng, T., Valentine, M., Gao, J., Pingel, J., & Yokoyama, W. (2009). Stability of Murine Cytomegalovirus Genome after In Vitro and In Vivo Passage Journal of Virology, 84 (5), 2623-2628 DOI: 10.1128/JVI.02142-09
2. Well asked, invisible non-existent person!
3. CASTRO, C., ARNOLD, J., & CAMERON, C. (2005). Incorporation fidelity of the viral RNA-dependent RNA polymerase: a kinetic, thermodynamic and structural perspective. Virus Research, 107 (2), 141-149 DOI:10.1016/j.virusres.2004.11.004

DIfferent strains of mice have different resistance to retrovirus infection. Some strains are highly resistant, others quite susceptible. At least some of this difference in susceptibility comes down to different expression levels of mouse APOBEC3: High expression of the gene gives good resistance to some retroviruses, low expression gives less resistance.

How come some strains have higher expression than others? Turns out that it’s because a retrovirus inserted in the APOBEC3 region of the genome of certain mouse strains, and that insertion cranks up expression of the APOBEC3.

We discovered that the mA3 allele in virus resistant mice is disrupted by insertion of the regulatory sequences of a mouse leukemia virus, and this insertion is associated with enhanced mA3 expression.  ((Sanville, B., Dolan, M., Wollenberg, K., Yan, Y., Martin, C., Yeung, M., Strebel, K., Buckler-White, A., & Kozak, C. (2010). Adaptive Evolution of Mus Apobec3 Includes Retroviral Insertion and Positive Selection at Two Clusters of Residues Flanking the Substrate Groove PLoS Pathogens, 6 (7) DOI: 10.1371/journal.ppat.1000974))

So perhaps low APOBEC expression allowed retrovirus infection, which led to insertion of the retrovirus genome, which increased APOBEC3 expression and provided resistance to further retrovirus infection.

]]> http://www.iayork.com/MysteryRays/2010/07/29/genetic-ironies-retrovirus-version/feed/ 0 http://www.iayork.com/MysteryRays/2010/07/26/quasispecies-thoughts/ http://www.iayork.com/MysteryRays/2010/07/26/quasispecies-thoughts/#comments Mon, 26 Jul 2010 10:15:30 +0000 iayork http://www.iayork.com/MysteryRays/?p=2488

Quasispecies theory predicts that slower replicators will be favored if they give rise to progeny that are on average more fit; these populations occupy short, flat regions of the fitness landscape … Flat quasispecies accept mutation without a corresponding effect on fitness … A flat quasispecies with an expansive mutant repertoire can explore vast regions of sequence space without consequence and is poised to adapt to rapid environmental change.

–Lauring, A., & Andino, R. (2010). Quasispecies Theory and the Behavior of RNA Viruses PLoS Pathogens, 6 (7) DOI: 10.1371/journal.ppat.1001005

(My emphasis)  RNA viruses in general form quasispecies because they have such high mutation rates.  Many (though by no means all) emerging infections are the result of RNA viruses. I’ve pointed before to aspects of viruses that might help them jump from species to species (for example, here — though this is a DNA virus, not an RNA virus, and I don’t think it runs in quasispecies — and the string of posts I link to inside that one).

One example Lauring and Andino point to is influenza virus hemagglutinin (HA). Influenza mutates very rapidly, of course, but most of the changes are harmful to the virus. But changes in HA seem to be very well tolerated. Since HA is a major target of the immune system, this property allows influenza to avoid the immune system without getting hit by defects in fitness associated with the immune evasion.  This is a contrast to, say, HIV (generalizing here! This isn’t always true). HIV within a patient undergoes constant changes to avoid the immune response, but many of these changes reduce the overall viral fitness. If you take the mutated HIV into an environment without that particular immune response, the virus quickly mutates back to its original, more-fit, form.

I’m not sure how we would assess, in advance, which viruses are more “flat” than others and that are therefore more able to adapt to new species, but it’s something to think about as we look at new viruses and new viral variants. I would be interested in SARS, for example — what happened to mutation tolerance as the virus adapted to humans? Was the virus that originally jumped into humans different in this was from the ones that normally infect bats? Not easy to measure, though.

I don’t have any particular insights into this, but it’s striking to me that there may be some parallel to the vastly worse 1918 pandemic.  Like 2009, the 1918 flu did virtually all its damage in the USA in less than a month, around October of 1918. (The difference, of course, was that in 1918 the virus killed far more of the people it infected.) In spite of the huge number of deaths that virus caused, though, it seemed to quickly recede into people’s memory as well.  I refer you to Alfred Crosby’s history of the outbreak (Amazon link), which is actually called “America’s Forgotten Pandemic: The Influenza of 1918″, for  a much more detailed discussion.

Is there something about these sort of explosive, but short-lived, outbreaks that lets them be easily replaced in peoples’ anxiety closet?

Hedskog, C., Mild, M., Jernberg, J., Sherwood, E., Bratt, G., Leitner, T., Lundeberg, J., Andersson, B., & Albert, J. (2010). Dynamics of HIV-1 Quasispecies during Antiviral Treatment Dissected Using Ultra-Deep Pyrosequencing PLoS ONE, 5 (7) DOI: 10.1371/journal.pone.0011345

The whole deep sequencing thing is going to profoundly change our knowledge of viral pathogenesis, as well as their ecology.

With highly mutation-prone viruses like HIV, hepatitis C virus, or influenza, our understanding of genome sequences has been based on the overall average genome — the average of a vast and diverse population. That average, that we’ve been calling the genome of these viruses, may not even exist as such, and certainly the minor variants that have been missed by traditional methods are also critically important, because they can explode out within a few days to take over the entire population, given the right set of circumstances. For example, if among those minor variants there are a few drug-resistant strains, then as soon as you treat the host, those variants may be able to take over.

In this paper, deep sequencing of people with HIV shows that drug-resistant variants do exist even before treatment, but they are normally very rare. They can take over during treatment with the particular drug, but when treatment is stopped they rapidly regress to rarity. This is presumably because the drug resistance makes the virus globally less fit (in the natural selection meaning of the term). When their more-fit brethren are destroyed by a drug these crippled, but drug-resistant, variants can grow out, but remove that selective pressure and the more wild-type versions take over once again.

As well as implications for treatment, this tells us something about viral reserves:

In most patients, drug resistant variants were replaced by wild-type variants identical to those present before treatment, suggesting rebound from latent reservoirs. ¹

1. Hedskog, C., Mild, M., Jernberg, J., Sherwood, E., Bratt, G., Leitner, T., Lundeberg, J., Andersson, B., & Albert, J. (2010). Dynamics of HIV-1 Quasispecies during Antiviral Treatment Dissected Using Ultra-Deep Pyrosequencing PLoS ONE, 5 (7) DOI: 10.1371/journal.pone.0011345

MHC class I is the target for cytotoxic T lymphocytes (CTL), which are generally believed to be pretty important in controlling viral infection. So when some viruses were shown to block MHC class I in cultured cells, it seemed pretty obvious that this would be a big benefit for the virus. You’d expect these viruses to be exceptionally resistant to CTL, for example.

But when people actually looked in animals (as opposed to in tissue culture), the ability to block MHC class I didn’t seem to do all that much. I’ve summarized some of those experiments here and here. For example, the MHC class I immune evasion genes in adenoviruses and in mouse cytomegalovirus (MCMV) didn’t show much effect on the actual infection at all.¹ Mouse herpesvirus 68 (MHV68) had shown an effect, but not at the time point that you might expect — not early after infection, when CTL are kicking in and clearing virus, but rather later on, during the latent phase.²

We all believed there must be a function, because viruses don’t hang on to genes for millions of years unless those genes are important,³ but I was starting to wonder if perhaps we were looking in the wrong places — whether any immune effects might be spillover from some other function, say. But, as I say, we’re starting to get confirmation that these things really are doing more or less what we’d expected all along.

A little while ago, Klaus Fruh and Louise Pickert showed a significant effect of MHC class I immune evasion in rhesus cytomegalovirus: without that ability new viruses couldn’t superinfect hosts that already carry the virus. ⁴ (I talked about it here.) It’s quite possible — though of course not certain until it’s actually tested — that this is also true for human cytomegaloviruses (which are very closely related to the rhesus version) and for mouse CMV (which are less closely related but in the same family). So now we have functional data for MHC class I immune evasion for representatives of two broad groups of viruses, the betaherpesviruses (the cytomegaloviruses) and the gammaherpesviruses (the MHV68 story).

Now there’s another paper⁵ showing a function for the MHC class I immune evasion ability of HIV (actually for SIV, but again it’s probably true for the closely-related HIV).

HIV has a gene, nef, that can block MHC class I expression. This has been shown in cultured cells, but understanding its relevance in actual infections has been difficult:

Although these data suggest that Nef-mediated immune evasion could play an important role in AIDS pathogenesis, there has been little direct evidence linking disease progression with MHC-I downregulation in vivo. ⁵

Obviously you can’t make a nef-less HIV and just throw it into people to see what happens. Even doing the experiment in monkeys with SIV is complicated by the fact that nef is very polyfunctional — as well as downregulating MHC class I, it also targets a number of other molecules.

But you can take advantage of natural variation, both in the virus and the host.  Nef isn’t equally effective on all MHC class I types, for one thing. As well, nef can develop mutations within the host.  It turns out that rapid disease progression correlates with the extent of MHC class I downregulation, whereas effects on other genes affected by nef (CD3 and CD4) didn’t correlate:

The extent of MHC-I downregulation on SIV-infected cells varied among animals …  the level of MHC-I downregulation on SIV-infected cells was significantly greater in the rapid progressor animals than in normal progressors.  … high levels of MHC-I downregulation on SIV-infected cells are associated with uncontrolled virus replication and a lack of strong SIV-specific immune responses.⁵

This is strictly a correlation study, so we can’t confidently say that MHC downregulation causes disease progression. Still, it’s an interesting finding, and perhaps one that can be followed up in human studies.

1. Gold MC, Munks MW, Wagner M, McMahon CW, Kelly A, Kavanagh DG, Slifka MK, Koszinowski UH, Raulet DH, & Hill AB (2004). Murine cytomegalovirus interference with antigen presentation has little effect on the size or the effector memory phenotype of the CD8 T cell response. Journal of immunology (Baltimore, Md. : 1950), 172 (11), 6944-53 PMID: 15153514
   Only slightly qualified by
   Lu, X., Pinto, A., Kelly, A., Cho, K., & Hill, A. (2006). Murine Cytomegalovirus Interference with Antigen Presentation Contributes to the Inability of CD8 T Cells To Control Virus in the Salivary Gland Journal of Virology, 80 (8), 4200-4202 DOI: 10.1128/JVI.80.8.4200-4202.2006
2. Stevenson, P., May, J., Smith, X., Marques, S., Adler, H., Koszinowski, U., Simas, J., & Efstathiou, S. (2002). K3-mediated evasion of CD8+ T cells aids amplification of a latent ?-herpesvirus Nature Immunology DOI: 10.1038/ni818
3. I will admit there’s a certain circular quality to this argument.  ”The gene must be important, because viruses don’t carry unimportant genes.  We know that, because this gene that they’ve hung on to must be important.”
4. Hansen, S., Powers, C., Richards, R., Ventura, A., Ford, J., Siess, D., Axthelm, M., Nelson, J., Jarvis, M., Picker, L., & Fruh, K. (2010). Evasion of CD8+ T Cells Is Critical for Superinfection by Cytomegalovirus Science, 328 (5974), 102-106 DOI: 10.1126/science.1185350
5. Friedrich, T., Piaskowski, S., Leon, E., Furlott, J., Maness, N., Weisgrau, K., Mac Nair, C., Weiler, A., Loffredo, J., Reynolds, M., Williams, K., Klimentidis, Y., Wilson, N., Allison, D., & Rakasz, E. (2010). High Viremia Is Associated with High Levels of In Vivo Major Histocompatibility Complex Class I Downregulation in Rhesus Macaques Infected with Simian Immunodeficiency Virus SIVmac239 Journal of Virology, 84 (10), 5443-5447 DOI: 10.1128/JVI.02452-09

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