Mystery Rays from Outer Space

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.[↩]

Sand rat, Psammomys obesus

Although a lot of viruses have ways of blocking recognition by T cells and NK cells, there’s not much known about the importance of these mechanisms in actual infections. That’s because the best-studied viruses in this class tend to be highly species-specific. So, for example, we don’t have good animal models for the human herpesviruses human cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, or Kaposi’s sarcoma herpesvirus. Herpes simplex virus does infect mice, but its immune evasion molecule ICP47 doesn’t work well in mice, so we’re no further ahead.¹

Immune evasion by all these viruses has been studied pretty extensively in cultured cells, but because they essentially only infect humans we only have circumstantial evidence for a role in vivo. Similarly, porcine, bovine, and equine herpesviruses encode immune evasion molecules, but pigs, cattle, and horses are not very convenient models for basic research either.Another major group of viruses, besides the herpesviruses, that are noted for immune evasion are the adenoviruses. However, only the human (and primate) adenoviruses contain the classical E3gp19k immune evasion molecule. There’s an animal model for human adenoviruses (the cotton rat) but as I pointed out the other day, there’s little evidence for an important function of CTL immune evasion in this model.

So what we need are small animal, and preferably lab mouse, models for infection with adenoviruses or herpesviruses that include immune evasion molecules. As far as we know, mouse adenoviruses don’t have T cell or NK cell immune evasion properties. That leaves us with mouse herpesviruses. Of the hundreds of known herpesviruses, six are known to be murid-specific (see the table at right), and three of those infect lab mice. One of those is totally obscure (there’s very little known about mouse thymic herpesvirus), leaving us with mouse cytomegalovirus and mouse herpesvirus 68 (MHV68). I’ve already commented on immune evasion by mouse CMV. The bottom line is that removing all known T cell evasion molecules from MCMV makes almost no difference to infection or latency; the one difference is that the virus persists longer and at higher levels in salivary glands. That may be important in virus transmission, but lacks a little oomph.

That leaves MHV68, and I’m pleased to say that there is actually some evidence that T cell immune evasion is important for this guy. (I’ve mentioned this in passing earlier, but it deserves its own post.) MHV68 uses a gene “mK3″ to attack MHC class I (MHC class I is recognized by cytotoxic T lymphocytes). ² In 2002, Philip Stevenson and Stacey Efstathiou made a mutant of MHV68 lacking mK3,³ and tested its ability to infect mice:

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 γ-herpesvirus. Nature Immunology DOI: 10.1038/ni818

In cultured cells, where there’s no immune system, the mutant virus grows exactly as well as wild-type MHV68. As well (more surprisingly) there was no difference in the initial virus clearance; the mutant virus and the parent were both cleared from the lungs of infected mice at the same rate, and were undetectable after about 13 days. However (finally!) there was a big difference in the amount of latency. The figure to the right shows latent wild-type (left panel) and mutant (right panel) MHV68, the black dots, in spleens of mice infected 13 days previously. What’s more, during the latent phase there was a better immune response to mutant MHV68; mice infected with the mutant virus had about twice as many CTL specific for MHV68.

The association of higher virus-specific CTL frequencies with lower viral loads suggested that CTLs were responsible for the elimination of DeltaK3 viruses during latency amplification.

Eliminating CTL from the mice removed the difference; in the absense of CTL, the mK3 knockout virus established latency just as well as the wild-type. This shows that effects on CTL, and probably not some other unknown function of mK3, are responsible for the difference.

So with the two and a half authentic models of infection (I count the herpes simplex virus one as a half because it’s more contrived than a natural infection) we have immune evasion molecules helping to establish latency (MHV68), helping to reactivate from latency (herpes simplex) and helping with persistence (MCMV) . In no case is there much effect on acute infection.

1. However, swapping in a different immune evasion molecule, that does work in mice, helps HSV reactivate from latency; see my previous post here. [↩]
2. mK3 is so named because it’s highly similar to a Kaposi’s sarcoma herpesvirus gene called “K3″; K3 is one of two KSHV genes that target MHC class I, but we don’t know much about KSHV infection. MHV68 probably isn’t a great model for KSHV, in spite of using a similar protein in immune evasion.[↩]
3. This virus was still able to down-regulate MHC class I to some extent, though, so there may be still other immune evasion genes in MHV68[↩]