Mystery Rays from Outer Space

Meddling with things mankind is not meant to understand. Also, pictures of my kids

February 24th, 2010

The deadliest, most awe-inspiring of the plagues

Yellow Jack flagMost of us don’t think much about yellow fever nowadays. There are still a couple hundred thousand cases, and some 30,000 deaths, each year, but almost none are in the first world. Out of sight, out of mind.

But this indifference is new. Until the beginning of the 20th century, yellow fever ran rampant, and was one of the most dreaded of all diseases. Epidemics of yellow fever in New York, Philadelphia, Memphis, and New Orleans killed tens of thousands. There’s a WHO manuscript [pdf link] on yellow fever that lists these and many more outbreaks – page after page of fine-print dates and deaths.

Those who have not lived between Cancer and Capricorn can well fail to conceive readily the sensation of numb, chill dreariness which steals on all hearts, when the news spreads, from mouth to mouth, that Yellow Jack has once more come. … at last it is admitted on the housetops, as well as whispered in the closet, that the deadliest, most awe-inspiring of the plagues of the equatorial regions has obtained admittance within our borders.

–“Yellow Jack“, in Cornhill Magazine, 18921

As I noted earlier, Carlos Finlay made the original suggestion that yellow fever was a mosquito-borne disease in 1881;2 in English, in 1886. 3 Walter Reed and his team confirmed this in 1900, and the discovery was seized on at once.

Mosquitoes in New Orleans, 1905 Yellow fever cases, New Orleans, 1905
“Shows the Distribution of the Principal Mosquitoes of New Orleans”.
Dark squares represent Stegomyia fasciata, the major carrier of yellow fever
Yellow fever cases in New Orleans, 1905.
“The infected blocks are most numerous in the old, Italian, quarter of the city.”

Yellow fever poster, New Orleans, 1905

(This post was mainly an excuse to post those maps.5  Click for larger versions.)

New outbreaks were checked with enthusiastic mosquito control.

In a few days with very little opposition, sixty to seventy thousand cisterns had been screened in order to prevent the breeding of the Stegomyia fasciata. Mosquito nets became more than ever the rule …

Yellow Fever Prophylaxis in New Orleans, 19055

The virus itself was was isolated in 1927 and the vaccine, made in 1937 by Max Theiler, turned out to be extremely effective; but even before that,  the understanding that mosquitoes were the carriers allowed great strides in reducing the disease.  Not just in the USA, but throughout the Americas:

Havana and Cuba freed from fever by Gorgas, who organized anti-mosquito measures, 1901-1902; example followed in Rio de Janeiro and Vera Cruz, 1903-1909; Panama Canal Zone successfully protected by same methods, 1904-1906 …  intensive campaign, 1918-1919, under Connor eliminated disease from Guayaquil, the chief endemic centre …

–“Yellow Fever in Retreat“, 19226

  1. Cornhill Magazine
    New Series, Vol. XIX, July to December 1892
    Smith, Elder, & Co.
    15 Waterloo Place, London []
  2. C. Finlay (1881). El mosquito hipoteticamente considerado como agente de trasmislon de la flebre amarllla An. de la Real Academia de ciencias med. de la Habana, 18, 147-169[]
  3. C. Finlay (1886). Yellow Fever, its transmission by means of the Culex mosquito Am. Journ. Med. Sci., 92, 395-409[]
  4. If I follow this right — I’m neither an entymologist nor an entomologist — Stegomyia fasciata was subsequently renamed Stegomyia aegypti, then Aedes agyptyi, and now (since 2005) is properly is officially called Stegomyia aegypti once again but usually, if not always, with “Aedes” in brackets to clarify. There was also, maybe, a point at which it was Stegomyia calopus, unless that was something else.[]
  5. Yellow Fever Prophylaxis in New Orleans, 1905
    Rubert Boyce
    April, 1906
    Published for the Committee of the Liverpool School of Tropical Medicine
    by Williams & Norgate
    14 Henrietta Street, Covent Garden, London[][]
  6. Current History
    A Monthly Magazine of the New York Times
    Volume XVI, April-September, 1922[]
February 22nd, 2010

Rabbits, virulence, history, and connections

Man chasing rabbit
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, 1894
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 Brodel, 1932)
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 [][][][][][]
February 16th, 2010

On predictability of influenza pandemics

Despite the fact that the recent pandemic was the best studied and recorded to date, the knowledge gained will probably have little predictive value for the next pandemic, either in qualitative or quantitative terms.

Communicable Diseases and Epidemics
Martin M. Kaplan
Bulletin of the Atomic Scientists, June 1960

Kaplan  was referring to the 1957-58 influenza pandemic, but the sentence could have been taken from many places in 2009. In any case, Kaplan was pretty much correct; the next three pandemics after that were quite different from the 1957-58 pattern, and quite different from each other.

February 13th, 2010

How many Americans are immune to H1N1?

Edit: I’ve updated the table to reflect the CDC’s numbers for age distribution of infection, which I didn’t see first time around.  Thanks to Marcello Pucciarelli for the link.  The original version of this post, containing my guesswork on the distribution, is still available here. Using the more accurate numbers has very little effect on my overall conclusions.

I’ve been expecting a resurgence of swine-origin influenza virus (SOIV) in North America for a while now, and it hasn’t happened. The virus is still out there, still infecting a few thousand people per week, but there hasn’t been a third large-scale wave of virus transmission. That’s different from the 1918 and 1957 pandemics (see here for details). What’s different this year?

Of course there are tons of things that are different this year, but I recently got around to doing something I should have done a while ago: Trying to estimate what proportion of Americans are now immune to SOIV. I’ve seen estimates that around 40% of the population should now be immune. I get roughly the same number (slightly higher, closer to 50%, because those estimates didn’t take into account pre-existing immunity to SOIV), but I think there’s an important point that might be missed in this: Most of the immunity might be clustered in the two most susceptible populations (children and the elderly), with two-thirds to three-quarters of them being immune.

There are three ways someone could be immune to SOIV. They could have been exposed to a related virus, some time in the past, and have developed a long-term immunity. They could have been infected with SOIV, somewhere in the first or second wave. Or, of course, they could have been vaccinated.

Numbers for each of those are available. They’re more or less approximate; not all the sources break down age groups in exactly the same way, for one thing, and I don’t have precise numbers for everything.1 I’ll try to flag places where I’m especially guessing, and it’s entirely possible that this I’ve made some obvious, basic mistakes in here, since this has been written in the interstices of cleaning our house for Chinese New Year (a Herculean task) and hosing down the kids to get them ready for the party at YongHui’s this evening.  If so, let me know and I’ll try to correct them.

We need to break immunity down by age, because pre-existing immunity to SOIV was strongly age-dependent. (That’s presumably why the virus was strongly biased to infecting younger people this year.) For the demographics of the US I’m using the 2008 census data. All this is summarized in the table below, and my explanations follow.

Age group Number Already immune Infected Vaccinated Vaccinated uninfected Number immune (low) Number immune (high) Percent immune (low) Percent immune (high)
0-18 years 82,640,086 3,305,603 19,000,000 30,576,831 25,838,759 48,144,362 52,882,435 58.3% 64%
19-64 years 182,549,922 10,952,995 33,000,000 38,335,483 32,570,355 76,523,350 82,288,479 41.9% 45.1%
65 and older 38,869,716 13,215,703 5,000,000 11,334,214 6,751,594 24,967,298 29,549,918 64.2% 76%
Totals 304,059,724 27,474,302 57,000,000 80,246,530 65,160,708 149,635,010 164,720,833 49.2% 54%

1. Pre-existing immunity. A paper in New England Journal of Medicine last year2 found that 4% of children, 6% of young adults, and 34% of older adults (born before 1950; I’m going with 65 years old as the dividing line just to make it easier to compare to other data) were already immune to H1N1. That’s more or less consistent with other studies I’ve seen, so let’s go with that.

2. Infection. The CDC estimates that somewhere over 55 million people in the US were infected in the first and second wave of SOIV, and gives approximate age distribution here. I’ve used the mid-point of their range estimates, so it’s possible that significantly more people were infected.  This works out to a quarter of US children being infected with SOIV,  which is broadly consistent with measures elsewhere — for example, a recent Lancet paper3 that estimated that about a third of children in the UK were infected.

3. Vaccination. The CDC’s Anne Schuchat’s Feb 5 press conference was very useful for this. The CDC has estimated that somewhere over 70 million people in the US have been vaccinated. Schuchat gave two further figures: Some 37% of children, and about 21% of adults have been vaccinated. These figures come from two different sources — a CDC survey and a Harvard survey respectively — so they may not be directly comparable, and I don’t know the breakdown in adults (young adults vs. elderly) but these figures do work out to about the right total, around 80 million people. Probably a little high, but not by much.

Another source of fuzziness is how much overlap there is between infected people and vaccinated people. It’s probably safe to say that most vaccinated people were not subsequently infected, but it’s quite possible that people were infected (perhaps with no symptoms, which we know happened quite frequently) and were subsequently vaccinated. 4 I’ve tried to adjust for this by assuming that half of infected people didn’t know it, and went on to get vaccinated, 5 as well as subtracting the proportion of people who were already immune (who presumably had no way of knowing that).  That’s the “Vaccinated uninfected” column.  Or, I’ve assumed  no overlap (just plain “Vaccinated”), to get an approximate range of immunity out there.

Including “Vaccinated uninfected” gives the “Number immune (low)”; assuming that all vaccinated were not infected gives the “Number immune (high)”.

And my conclusions are that:

  • Over half the US population as a whole is now immune to the new SOIV.
  • As many as three-quarters of the elderly and two-thirds of the children — the most important populations as far as flu is concerned — may be immune.
  • Between a third and about half of this immunity was due to vaccination.

That level of immunity is probably enough to impact virus transmission drastically. In the early waves, if a child was infected then virtually every child she contacted in school or on the playground would be susceptible. Now only one in three of them are potentially infectable. I’ll have to spend some time looking at the models of influenza spread but I think that considering that the SOIV was not spectacularly infectious anyway, this level of population immunity is easily enough to prevent the third winter wave of disease I was expecting to see.

(I am particularly curious about modeling the impact of vaccination. Without vaccination would there have been a third wave? My guess is that there would have been, but that’s just a guess.  Update for clarification: Vaccination rates were highest in children. Without vaccination only about 25% of children would be immune — vaccination therefore doubled or tripled the amount of immunity in this critical population, and I think SOIV would have resurged in schools in winter without this intervention.)

What’s more, this level of immunity, especially in the apparent absence of the usual seasonal flu strains, has important implications about influenza over the next few years, but this post is already too long, so maybe I’ll talk about that some other time.

  1. Probably quite accurate numbers are available, but not to me. Or, at least, not without a lot more work.[]
  2. Hancock, K., Veguilla, V., Lu, X., Zhong, W., Butler, E., Sun, H., Liu, F., Dong, L., DeVos, J., Gargiullo, P., Brammer, T., Cox, N., Tumpey, T., & Katz, J. (2009). Cross-Reactive Antibody Responses to the 2009 Pandemic H1N1 Influenza Virus New England Journal of Medicine, 361 (20), 1945-1952 DOI: 10.1056/NEJMoa0906453[]
  3. Miller, E., Hoschler, K., Hardelid, P., Stanford, E., Andrews, N., & Zambon, M. (2010). Incidence of 2009 pandemic influenza A H1N1 infection in England: a cross-sectional serological study The Lancet DOI: 10.1016/S0140-6736(09)62126-7[]
  4. This is another significant potential source of error, I think.[]
  5. That is, I’ve subtracted that proportion of people from the vaccinated totals.[]
February 12th, 2010

Leprosy and the Silk Road

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.

Phylogeography of leprosy

Pillars are located on the country of origin of the M. leprae sample … The gray arrows indicate the migration routes of humans, with the estimated time of migration in years shown. The red dots indicate the location of the Silk Road in the first century.” 3 (Click for a larger version)

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 [][][]
February 10th, 2010

A scarifying story

God of Smallpox
Sopona, the Yoruba god of smallpox

A while ago I listed a number of reasons why smallpox was eradicated, whereas other diseases haven’t been (yet). One of the reasons was that the vaccine against smallpox1 is so effective. Vaccinia immunization induces immunity for an extraordinarily long time — memory immune responses have been shown for up to 60 years after vaccination.

So why is vaccinia such an effective vaccine? Part of it is that vaccinia is a live virus: It replicates after you’re inoculated (so there’s lots of antigen there), and it stimulates the innate immune response (which is geared toward detection of live viruses, among other things). (The yellow fever vaccine is another live virus vaccine that’s also famous for inducing long-term immunity.) Vaccinia is also a large virus that has a lot of antigens available, so that there are lots of different modes of immunity triggered. That is, both B cell (antibody-based) immunity, and broad T cell-based immunity, are likely to be present and to have lots of different targets.

A recent paper2 suggests that the route of vaccination is also important. Unlike most vaccines, which are given by intramuscular (e.g. influenza vaccine) or subcutaneous (e.g. yellow fever) injection, or orally (the live polio vaccine), vaccinia is delivered by scarification — scraping the most superficial layers of the skin. I don’t think this was the result of deliberate comparisons — scarification was the traditional method, and it was easy and convenient. Before 1967:

A scratch about 5 mm long was made in the skin with a needle, a lancet or a small knife and the vaccine suspension was rubbed into the site. A single cut or cross cuts were made, in 1 , 2 or 4 different sites. This was essentially the same method as had been used for variolation in Europe during the latter part of the 18th century. ((Smallpox and its Eradication (Chapter 7). F. Fenner, D. A. Henderson, I. Arita, Z. JeZek, I. D. Ladnyi. World Health Organization, Geneva, 1988))

Later, a bifurcated needle was used:

Experiments soon showed that the multiple puncture method, in which the bifurcated needle was held at right angles to the skin, which was then punctured several times with the prongs, was very efficient and very easy even for an illiterate vaccinator to learn. It became the standard method of vaccination throughout the world. 3

Scarification was a simple and convenient way to deliver the vaccine.  It turns out that scarification isn’t just a convenience, it’s the most effective way to get immunity:

VACV immunization via s.s. [skin scarification], but not conventional injection routes, is essential for the generation of superior T cell–mediated immune responses that provide complete protection against subsequent challenges.2

Langerhans cells J Dermatol Sci
Langerhans cells in the skin4

This includes protection against respiratory-spread disease, not just skin infection. My first thought was that this is probably simply because the vaccinia virus replicates better in the skin than by intramuscular injection, but the improved immunogenicity is also seen with a non-replicating version of vaccinia, “MVA”. 5

My next thought is that Langerhans cells are probably part of the reason. Langerhans cells (see the figure to the right) are a subset of dendritic cells, probably extremely good at triggering immunity, that form a dense network under the skin, and probably act very efficiently at filtering skin-delivered antigen and delivering it to the immune system.

Also, the fact that the skin is damaged in the process evokes Polly Matzinger’s “danger” concept of immune stimulation.

At any rate, something, even if we don’t know exactly what, about scarification leads to better immunity, at least for vaccinia virus. That’s useful to know. Having said that, I’m not quite sure why this paper appeared in Nature Medicine, a very high-impact journal — the mechanism wasn’t shown at all clearly, and this isn’t the first time that the general observation has been made:

This study strongly indicated that, although less reactogenic, vaccinia vaccine administered im [intramuscularly] at a dose of 105 pfu fails to induce an immune response comparable to that elicited by standard scarification. 6

Even more broadly, the skin inoculation concept has been shown to lead to high immunogenicity in other systems; for example, it was shown a couple of years ago that yellow fever vaccine is more immunogenic when delivered intradermally than when given by its conventional subcutaneous route:

Intradermal administration of one fifth of the amount of yellow fever vaccine administered subcutaneously results in protective seroimmunity in all volunteers. 7

BIfurcated needle
Bifurcated needle used for smallpox vaccination

(I do have to add that apparently scarification — which is much easier than intradermal injection — does not work for yellow fever, based on some experiments in the 1950s.8 I haven’t read those papers myself, though. I’d be interested to see if the bifurcated needles used in the late 1960s and on for vaccinia might be more effective for the yellow fever vaccine.)

Anyway, seeing this in at least two instances9 makes it seem possible that it’s a general effect. If skin administration enhances immunogenicity, perhaps this is a way of extending limited vaccine stocks in an emergency.

  1. That is, vaccinia virus[]
  2. Liu, L., Zhong, Q., Tian, T., Dubin, K., Athale, S., & Kupper, T. (2010). Epidermal injury and infection during poxvirus immunization is crucial for the generation of highly protective T cell–mediated immunity Nature Medicine, 16 (2), 224-227 DOI: 10.1038/nm.2078[][]
  3. Smallpox and its Eradication (Chapter 11).  F. Fenner, D. A. Henderson, I. Arita, Z. JeZek, I. D. Ladnyi.  World Health Organization, Geneva, 1988[]
  4. MAHNKE, K., JOHNSON, T., RING, S., & ENK, A. (2007). Tolerogenic dendritic cells and regulatory T cells: A two-way relationship Journal of Dermatological Science, 46 (3), 159-167 DOI: 10.1016/j.jdermsci.2007.03.002[]
  5. At any rate, it’s claimed to be non-replicating, but I don’t remember seeing it formally shown that MVA doesn’t replicate, even temporarily, in the skin. Anyone know if this has been tested?[]
  6. Immunologic Responses to Vaccinia Vaccines Administered by Different Parenteral Routes Author(s): David J. McClain, Shannon Harrison, Curtis L. Yeager, John Cruz, Francis A. Ennis, Paul Gibbs, Michael S. Wright, Peter L. Summers, James D. Arthur, Jess A. Graham Source: The Journal of Infectious Diseases, Vol. 175, No. 4 (Apr., 1997), pp. 756-763[]
  7. Roukens, A., Vossen, A., Bredenbeek, P., van Dissel, J., & Visser, L. (2008). Intradermally Administered Yellow Fever Vaccine at Reduced Dose Induces a Protective Immune Response: A Randomized Controlled Non-Inferiority Trial PLoS ONE, 3 (4) DOI: 10.1371/journal.pone.0001993[]
  8. Ann Trop Med Parasitol. 1953 Dec;47(4):381-93.  Vaccination by scarification with 17D yellow fever vaccine prepared at Yaba, Lagos, Nigeria.CANNON DA, DEWHURST F.

    Am J Hyg. 1952 Jan;55(1):140-53.  A preliminary evaluation of the immunizing power of chick-embryo 17 D yellow fever vaccine inoculated by scarification.DICK GW.[]

  9. And I’m pretty sure I’ve seen at least one other example, but I’m blanking on the details[]
February 5th, 2010

On the magic bullet virus

Vesicular stomatitis virus (VSV), like the related rabies virus, is a bullet-shaped virus.  Hong Zhou has just added VSV to his collection of cryo-electron microscopy virion structures,1 and as always with viruses, it’s just gorgeous.

Structure of VSV by cryo-electron microscopy VSV structure and assembly
“Architecture of the VSV virion. … A montage model of the tip and the cryo-EM map of the trunk”1 “A plausible process by which the nucleocapsid ribbon generates the virion head, starting with its bullet tip.”1

  1. Peng Ge, Jun Tsao, Stan Schein, Todd J. Green, Ming Luo, & Hong Zhou (2010). Cryo-EM Model of the Bullet-Shaped Vesicular Stomatitis Virus. Science, 327 (5966), 689-693 DOI:10.1126/science.1181766[][][]
February 3rd, 2010

Tumors as ecosystems

Park et al JCI 2010 Fig 2
Clonal evolution during in situ to invasive breast carcinoma progression1

What’s a tumor?

In some ways, that’s a bad question (never mind the answer) because it implies that a tumor is a single thing. But we know that’s not true. A tumor, by the time we can detect it, is a collection of many cells, at least billions of them, and those cells are not all the same. I’m not even talking about the normal cell types that are incorporated into a tumor (things like blood vessels and support cells). Even cells that are unambiguously cancerous are very different within a tumor. And of course, that’s important for the things we’re most interested in, prognosis and treatment, because it’s not the average tumor cell that we’re most concerned about, it’s the subset of tumor cells that are most resistant to treatment, or that are most aggressive.

The development of this variation is really fundamental to how we understand tumor formation and tumor growth. Cancerous cells don’t just appear, fully ready to metastasize and grow. What happens is that a normal cell mutates slightly and gains a little advantage. Most of its progeny stay like that, but one of them mutates again and changes a little more, and then one of that cell’s progeny mutates again, and so on. It probably takes at least a half-dozen mutations, over many cell generations, before a normal cell has progressed through to a detectably cancerous cell.  (I’ve talked about this before, here.)

Also, since truly normal cells simply don’t mutate that many times — there are too many checks and repair systems to allow a half-dozen mutations to accumulate in a single human’s2 lifetime — one of the mutations is probably in the check/repair system, turning the cancerous pathway into a mutator pathway as well.

So we expect tumors to be made up of many different cell types, and this is indeed what we see:

With rare exceptions, human malignancies are thought to originate from a single cell, yet by the time of diagnosis, most tumors display startling heterogeneity in cell morphology, proliferation rates, angiogenic and metastatic potential, and expression of cell surface molecules. 1

So how diverse are tumors?

That’s been a hard question to answer, because you’d need tools to look at individual cells, and you’d also need some way of expressing that diversity. A recent paper1 looked at diversity in breast cancer using some individual-cell tools, which I’m not going to discuss, and took an interesting approach to describing the variability:

… we applied diversity measures from the ecology and evolution sciences to our copy number data. These diversity measures estimate the number and distribution of species in a certain geographical area or environmental niche. In our context, a species is a cancer cell population … Hence, a region of a tumor containing cancer cells with 3 different copy number ratios is interpreted to contain 3 distinct “species.” 1

They suggest that this way of describing tumors could be a useful aid to prognosis and to predicting response to therapy, offering a quantitative description of tumor variability (which might correlate with the tumor’s potential for spread and escaping treatment).

I hadn’t thought of tumors as ecosystems before, but I wonder if the analogy could be taken further by considering, say,cytotoxic T lymphocytes as predators …

  1. Park, S., Gönen, M., Kim, H., Michor, F., & Polyak, K. (2010). Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype Journal of Clinical Investigation DOI: 10.1172/JCI40724[][][][]
  2. let alone a mouse’s lifetime[]
February 1st, 2010

Virus discovery by jigsaw puzzle

Jigsaw (Wellcome Images)

Every so often — not often enough — I run across a paper that’s so ridiculously ingenious that it just makes me laugh with pleasure.

Ladies and gentlemen, a round of applause, please, to Shou-Wei Ding, of the Center for Plant Cell Biology at UC Riverside, for his Rube Goldberg-esque brilliant technique for identifying new viruses. 1

Background: Small interfering RNA (siRNA, 2 or RNAi) is pretty well known nowadays, especially since three of its discoverers were given Nobel Prizes a few years ago. These small RNAs are found in most eukaryotes — plants, insects, worms, as well as birds and mammals. In fact, siRNA was first discovered in plants and then was widely used in insects and worm research well before it was shown in mammals. In mammals, we tend to think of small RNAs as having regulatory functions. In plants, insects, and worms, there are certainly regulatory siRNAs, but siRNAs are also used as an important part of their antiviral immune response. (Unsurprisingly, plant and insect viruses themselves have defenses against these siRNAs, by producing anti-siRNA genes.)

siRNAs are small (duh), maybe 20-30 bases long. Without going into mechanisms more than necessary, they recognize specific sequences of their target RNA (by being complementary to the target). When they bind to their target, they cause that target RNA to be chopped up into small pieces. Some of those small pieces can then act as new siRNA and the cycle continues.

Ding’s group reasoned that (1) if insect viruses are being attacked by siRNA, and (2) the viruses are then chopped up into new siRNA, then (3) all you have to do is piece together the siRNA, to recover the virus sequence.

Seems sort of obvious now that I put it together that way, but I would have said that there’s no way it would work — not enough coverage, I would have said, you’d only see a tiny fraction of the genome. Even if there was enough coverage, how would you find the virus pieces in the huge pool of other siRNAs? And even if you could do that, how would you piece them together? It would be like a jigsaw puzzle where every piece was just a tiny snippet of blue sky.

But, wonderfully, it actually worked for Ding’s group. Not only did they identify viruses that they knew should be there, they also pulled out five brand-new viruses out of their insect cells:

In this study, we found that viral small silencing RNAs produced by invertebrate animals are overlapping in sequence and can assemble into long contiguous fragments of the invading viral genome from small RNA libraries sequenced by next-generation platforms. Based on this finding, we developed an approach of virus discovery in invertebrates by deep sequencing and assembly of total small RNAs (vdSAR) isolated from a host organism of interest. Use of this approach revealed mix infection of Drosophila cell lines and adult mosquitoes by multiple RNA viruses, five of which were previously undescribed.

Ding 2010 virus genome assembly
“Virus discovery in OSS cells by viral genome assembly from sequenced viral piRNAs of 25–30 nucleotides in length after viral siRNAs were removed.”

The ability to piece these tiny fragments together is a spin-off of the new genome sequencing platforms, which by their nature make very short reads that have to be computationally stitched back together. Most of these new platforms make slightly longer fragments than siRNA size, but they’re in the same ballpark and I guess the same approaches work.

As far as the coverage, they didn’t find 100% of the genomes, but they got a really surprisingly high fraction back — 80% to 95% or more of the various viruses.

Not only that, they got really new viruses:

As a result, none of the four viruses could be assigned into an existing virus genus. This suggests that vdSAR is capable of discovering viruses that are only distantly related to known viruses.

Giving credit where it’s due, another group recently, and independently, used a similar approach to identify viruses in sweet potatot plants3 but I didn’t notice that article until Ding pointed it out.

Whether or not this technique proves useful in the long run, it’s just so ingenious that I want it to succeed.

  1. Wu, Q., Luo, Y., Lu, R., Lau, N., Lai, E., Li, W., & Ding, S. (2010). Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs Proceedings of the National Academy of Sciences, 107 (4), 1606-1611 DOI: 10.1073/pnas.0911353107[]
  2. I’m going to call all of the various types of small RNAs “siRNA” here, but that’s just shorthand, there are different subclasses that I won’t go into[]
  3. Kreuze, J., Perez, A., Untiveros, M., Quispe, D., Fuentes, S., Barker, I., & Simon, R. (2009). Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: A generic method for diagnosis, discovery and sequencing of viruses Virology, 388 (1), 1-7 DOI: 10.1016/j.virol.2009.03.024[]