Virology pop quiz: Answers

30 July 2009

BaculovirusA few weeks ago I asked readers to find the errors in the following statement concerning an experimental influenza vaccine produced by Protein Sciences which involves synthesis of the viral HA protein in insect cells.

They warned that the virus could mutate during the southern hemisphere’s flu season before returning north in a more lethal form in autumn, in a pattern similar to that seen in the deadly 1918 flu pandemic, which claimed an estimated 20 to 50 million lives around the globe.

The CDC (Centers for Disease Control and Prevention) sent us a dead virus, which is perfectly safe, and then we extracted genetic information from that virus.

The statement ‘in a pattern similar to that seen in the deadly 1918 flu pandemic’ is wrong. There is no evidence that mutation led to the emergence of a ‘more virulent’ virus that caused more severe disease in the fall of 1918. The only virus available to study was reconstructed from material obtained in November 1918. The first influenza virus was not isolated until 1933. The idea that a more virulent virus emerged in the fall has nevertheless become firmly established – without any scientific evidence to support the hypothesis. See “Riding the influenza pandemic wave” for more information.

The second problem is the statement that CDC sent the company a dead virus. Viruses are not living, so they cannot be killed. What the company received is an inactivated virus which cannot replicate in cells. There are many ways to inactivate viral infectivity, including heat, ultraviolet radiation, or treatment with chemicals such as formalin.

For extra credit I asked readers to critique the following statement:

Protein Sciences’ technology is also safer “because these caterpillars don’t have any association with man or other animals, so there’s no chance for their cells to learn how to propagate human viruses,” Adams told AFP.

What exactly was the spokesman trying to say? That there is no chance that influenza virus will replicate in insect cells? That’s impossible to say. The fact that ‘caterpillars don’t have any association with man’ is irrelevant (I’m not an entomologist, but I don’t believe that statement is correct). It’s possible that the virus could be adapted to grow in insect cells in the laboratory. And of course, cells don’t ‘learn’ how to propagate viruses. When viruses are selected for growth in new cells – a process that we call expanding the tropism of the virus – changes in the viral genome are usually responsible.

Parenthetically, there is a better way to make an influenza virus vaccine in insect cells – by synthesizing virus-like particles. When the influenza viral HA, NA, and M1 proteins are made in insect cells, virus-like particles are produced that lack the viral genome. These have been shown to be immunogenic in ferrets, and are capable of inducing a protective immune response.

Ross, T., Mahmood, K., Crevar, C., Schneider-Ohrum, K., Heaton, P., & Bright, R. (2009). A Trivalent Virus-Like Particle Vaccine Elicits Protective Immune Responses against Seasonal Influenza Strains in Mice and Ferrets PLoS ONE, 4 (6) DOI: 10.1371/journal.pone.0006032

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  • phytosleuth

    “Protein Sciences’ technology is also safer “because these caterpillars don’t have any association with man or other animals, so there’s no chance for their cells to learn how to propagate human viruses,” Adams told AFP.”

    Yes, that's a humdinger of an unclear statement. Caterpillars on my garden plants aside, plenty of insects are virus vectors to both plants and people. Some of the most dangerous viruses in fact. Bacteria too, but that's another story. Whether insects propagate human influenza virus, I don't think anyone has found this yet. Not sure they are looking.

    In any case, one interesting fact is that insect hormones DO in fact have a biological effect in humans. We don't molt, thank goodness (actually I wouldn't mind right now though). The bioactivity of ecdysteroids in humans causes an adaptogenic effect. Adaptogenic compounds (usually from plants) support the ability of the body to adapt to stress. See Adaptogens in Wikipedia. Just to make things more confusing, plants synthesize insect hormones. It's thought this is a defensive strategy to cause endocrine (molting) disruption in insects that eat plants. Oh, and they eat plants to get the starter compounds to make their molting hormones.

    It's all a very interesting symbiotic circle.
    http://ecdybase.org/
    http://joe.endocrinology-journals.org/cgi/conte…

  • rodrose

    Extremely valuable information for journalists (I'm one) covering the H1N1 outbreak — Thanks. I especially appreciate the Influenza 101 posts.

  • h1n1_watcher

    >There is no evidence that mutation led to the emergence of a ‘more virulent’ virus that caused more >severe disease in the fall of 1918.

    Thanks for pointing this one out again.

    In the picture painted by the media it is presented almost as a given that the pandmics 'secod wave' in the fall will be much more severe while implying this will be due to an increase in pathogenicity of the virus itself.

    However, applying 'Occams Razor' principle to this phenomenon it is much more likely that in 1918 the increase in severity was simply due to the much higher number of total infections in the fall (due to environmental conditions such as low temperatures and humidity favoring virus spread ) rather than the alleged genetic changes towards more pathogenicity in the virus genome itself.

    BTW.: contrary to popular belive such marked changes in virus pathogenicity during successive waves have not consistently been observed in neither the 1957 nor the 1968 pandemics.
    (For example in 1968 the second wave was more severe in some countries wheras it was just the other way round with a more severe first wave in other countries [1] which again suggests reasons other than genetic chnages of the virus itself for the difference in severity accross different pandemic waves)

    ——————-
    [1]Viboud C, “Multinational impact of the 1968 Hong Kong influenza pandemic”

  • Brian Hanley

    Hi Vincent,

    A contrarian view here.

    It is in fact true that the probability of a virus crossing over from insects to mammals is quite low, although it probably can happen. (There is a speculation that Ebola Zaire may have an insect host as part of its normal life cycle, but this is highly speculative and not established. And viruses that cross species are, for evolutionary reasons, most likely to have extremely high mortality for evolutionary reasons.) The reason for lower probability is differences in attachment sites on cell membrane surfaces. So I'm not particularly fussed about that. It's a bit of marketroid bluffery, yes, but not entirely wrong, just a partial. To be complete they should have said it was very low, but if it ever did happen it could be devastatingly deadly. :-) (But the marketing folks would be horrified to be so complete, no?)

    The matter of evolution of higher virulence is still being discussed, but it comes straightfrom Darwinian evolutionary theory and epidemiology shows it as a pattern in influenza epidemics. I would refer the reader to the works of Paul Ewald who has done excellent work in the area of evolutionary adaptation of disease to hosts with quite a few diseases. http://en.wikipedia.org/wiki/Paul_W._Ewald . In a nutshell, microorganisms optimize their reproduction. The limit on that is vectoring between hosts. If the organism becomes too virulent then it kills its host more rapidly than it can spread and that strain dies out, so there is a balance that can be calculated. (For the mathematically minded, an equation can be derived from that description. A gold star to anyone who does so without looking it up.) Much of the evidence is from epidemiology and modeling, but taken in total it is quite convincing. Generation times are a bit fuzzy in viruses, but still amenable to modeling and very short.

    So in my view, of course viruses adapt to their hosts in time spans on the order of flu seasons. There is very respected literature on the 1918 epidemic in specific and excellent modeling efforts regarding this. So I take the side that adaptation to increased virulence (depending on vectoring conditions) is true until proven false. If the virus is passed from host to host rapidly in high inoculum, the more virulent strains will rapidly take over to the limit of the rate of passage from host to host. (i.e. There are more ways to prove something than to catch the live virus in the lab.) I'm traveling right now, so I don't have cites at my fingertips, but it would be worth inviting Paul Ewald to speak.

    The “dead virus” note is questionable. However! There is such a thing as an inactivated influenza virus that could be loosely called “dead”, since the virus is enveloped with the host cell membrane and depends on that membrane being intact to be infectious. Influenza viruses are probably deactivated in aerosol by osmotic absorption of essentially distilled water in humid air since they contain physiological salt concentrations inside. Pop the envelope and the virus is “dead.” And – it is completely possible to make cDNA from such a “dead virus”. Of course, I find it pretty darn unlikely that CDC would ship the virus deliberately in that form. They might have, but lack of infectiousness would have to be established by proper tests that aren't worthwhile. So, for all practical purposes that statement is, indeed, likely rubbish.

    Cheerio. :-)

  • h1n1_watcher

    >The limit on that is vectoring between hosts. If the organism becomes too virulent then it kills its >host more rapidly than it can spread and that strain dies out, so there is a balance that can be >calculated.

    This is, of course, true.

    But I still do not see the reason why there should be an evolutionary path towards higher pathogenicity.

    For the optimum reproduction rate is obviously reached if the virus both maximizes transmissibility while at the same time *minimizing pathogenicity*.

    The empirical evidence of this can be seen in the fact that the most successful human viruses (in the sense of reproduction) are probably the plain rhino viruses (common cold viruses) that are both extremely well transmissible while causing very mild disease.

    BTW.: not only do viruses adapt to the human population but, as can be obsereved during the periods following influenza pandemics, the human population co-evolutionarily adapts to the new virus, too. (That's why the death rate among young & otherwise healthy people is usually highest during the first waves of a pandemic and diminishes during later year's waves, simply because those most genetically susceptible to the virus have already died during the first wave)

  • Brian Hanley

    Actually, evolution simply says that the organism will maximize its reproduction, because the best reproducers will dominate. Hardy Weinberg shows just a few iterations with a small difference will rapidly go to population dominance. So, there is actually no reason at all why a disease cannot evolve to maximize its reproduction by killing its hosts and even wiping out an entire population of hosts. The only thing necessary is that the disease must continue to be vectored from host to host well for those most virulent forms. Myxomatosis is rabbit influenza, and it wiped out rabbits in australia until the population go t too sparse to propagate it. (99% mortality in total population)

    Please see Ewald's work. He has documented evolution of higher virulence in as little as 6 weeks simply due to differences in vectoring of the disease from host to host. I can't give an entire lecture here, but that's it in a nutshell.

    Regarding your second point it is incorrect. Yes, humans co-evolve with diseases, but over long time scales, certainly not in a matter of years. It takes multiple generations. Human generations are long. As I said above, what a generation is in influenza is a bit fuzzy, but it is on the scale of days. So your understanding of coevolution phenomenon is incorrect.

  • h1n1_watcher

    Brian, I actually agree with most points

    >So, there is actually no reason at all why a disease cannot evolve to maximize its >reproduction by killing its hosts and even wiping out an entire population of hosts.

    I Agree that such developments can not be excluded (as you say, nothing is excluded in evolution 'per se' if it only increases the reproduction factor) But this is obviuously not a *stable* evolutionary 'strategy' sustainable for longer periods of time or larger host populations. It rather represents a 'dead end' or an accident of (co-)evolution …

    So the general trend in co-evolution over long time spans will lead to either extinction of one organism or a both organisms being better adapted to each other in the long term.

    Again consider the most 'evolutionary succesful' (i.e. most widespread, most prevalent) human virus: the common cold (rhino) virus. It is almost perfectly adapted to the human population without causing anything but a very mild desease.

    >Yes, humans co-evolve with diseases, but over long time scales, certainly not in a >matter of years. It takes multiple generations. Human generations are long. As I said >above, what a generation is in influenza is a bit fuzzy, but it is on the scale of days.

    Again, I fully agree. I do think, however, that the effect I mentioned (decreasing severity in the years/decades follwing major pandmics) could be viewed as a one-generation effect of the selective pressure exerted by the virus on the human population. (i.e. those most genetically susceptible to the virus 'die out' during the early waves)

    >Please see Ewald's work. He has documented evolution of higher virulence

    ok, I will do this (thanks for the link) … sounds interesting.

    ————————
    P.S.: english is not my first language so maybe some formulations of mine could be mistakable or a bit cumbersome.

  • h1n1_watcher

    Brian, I actually agree with most points

    >So, there is actually no reason at all why a disease cannot evolve to maximize its >reproduction by killing its hosts and even wiping out an entire population of hosts.

    I Agree that such developments can not be excluded (as you say, nothing is excluded in evolution 'per se' if it only increases the reproduction factor) But this is obviuously not a *stable* evolutionary 'strategy' sustainable for longer periods of time or larger host populations. It rather represents a 'dead end' or an accident of (co-)evolution …

    So the general trend in co-evolution over long time spans will lead to either extinction of one organism or a both organisms being better adapted to each other in the long term.

    Again consider the most 'evolutionary succesful' (i.e. most widespread, most prevalent) human virus: the common cold (rhino) virus. It is almost perfectly adapted to the human population without causing anything but a very mild desease.

    >Yes, humans co-evolve with diseases, but over long time scales, certainly not in a >matter of years. It takes multiple generations. Human generations are long. As I said >above, what a generation is in influenza is a bit fuzzy, but it is on the scale of days.

    Again, I fully agree. I do think, however, that the effect I mentioned (decreasing severity in the years/decades follwing major pandmics) could be viewed as a one-generation effect of the selective pressure exerted by the virus on the human population. (i.e. those most genetically susceptible to the virus 'die out' during the early waves)

    >Please see Ewald's work. He has documented evolution of higher virulence

    ok, I will do this (thanks for the link) … sounds interesting.

    ————————
    P.S.: english is not my first language so maybe some formulations of mine could be mistakable or a bit cumbersome.

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