Amboceptor

Microbiological, virological, bacteriological, immunological, medical, epidemiological, historical, anecdotal

Tag: vaccinia

Data update: Destroyed by freezing does not equal alive

In the experiments reported in the present paper a number of active agents, some undoubtedly living, others equally unquestionably not living, and still others of a doubtful nature, were subjected to repeated freezing (-185°C.) and thawing. By these tests it has been possible to determine that mere destruction or inactivation of a substance cannot be accepted as proof that it existed in a living state.

In 1926, legendary virologist / bacteriologist Thomas M. Rivers addressed the still-extant question “Are viruses alive?” through the simple experimental method of freezing them over and over. The paper in question came out in the Rockefeller Institute house publication Journal of Experimental Medicine (vol.45[1]: pp. 11-21). Not exactly a gripping title.

repeated-freezing-rivers

What kind of data is in here? Well, for most of the graphs they take an “active agent” (either bacteria, virus, or enzyme), freeze-thaw it “as often as desired”, and then see if it’s still active. The data is pretty straightforward, with a separate section for colon bacilli, a section for Virus III, a section for “a bacteriophage lytic in colon bacilli”, and so on. But it’s never summarized in a way that compares the different “agents” to each other. Let’s try to do that.

The low temperature (-185°C.) used in the experiments to be reported was obtained by means of commercial liquid air which was transported from the plant to the laboratory in Dewar flasks. Desired amounts of the air were transferred to deep Dewar beakers where small amounts of the substances to be frozen, enclosed in Noguchi tubes, were completely immersed for several minutes. After the substances had been completely frozen they were quickly thawed in tap water (16-18°C.).

What does “active” mean for these various substances? How did they determine that freeze-thawing had destroyed the substance’s activity? This was different for each substance.

rivers-activity

Bacteria can be measured the same way we measure them now, by making serial dilutions and plating each dilution on agar, then seeing how many colonies grow. Bacteriophage can be measured the same way, by first making a “lawn” (agar plate fully covered with bacteria) and applying different dilutions of bacteriophage, then seeing how many “plaques” (holes in the lawn) are formed by the phage killing the bacteria.

Complement can be measured by seeing how long it takes to destroy “given amounts of red blood cells in the presence of a great deal of amboceptor“.

Trypsin can be measured somehow, Dr. Rivers doesn’t specify except by saying that his colleague Dr. Northrop took care of that part of the study. Nowadays Dr. Northrop would be a co-author. But the paper would then have to list Rivers as both the first author and the last author somehow, since Northrop didn’t do enough to merit either status.

Anyway, to find “details of the technic” we are directed to a paper by Northrop and Hussey (1923), showing a very clever method by which a solution of gelatin is exposed to trypsin, and at different timepoints the gelatin’s viscosity is measured.

northrop-graph

And how do you measure viscosity? With a viscosimeter.

northrop-viscosimeter

I’m having trouble understanding sentences like “The gelatin-water time ratio was approximately 3”, but the point is that you can measure the amount of trypsin by measuring how fast it turns gelatin into runny gelatin. Nowadays you would use a colorimetric assay, in which trypsin cuts the protein that has some sort of colored label attached to it, and you would measure how much colored label gets released into solution.

* * *

Finally, the three mammal viruses.

“Vaccine virus” and “Virus III” are both introduced to the skin of rabbits, probably by scarifying and then rubbing the virus into the scratches. They look for a “virus reaction” in the skin surrounding where the virus was inoculated, and measure this semi-quantitatively based on how bad of a sore forms. I hope they aren’t simply measuring the immune response to the inoculation, because even killed virus should produce some immune response.

“Vaccine virus” is basically what we now call vaccinia virus. “Virus III” is a more interesting question.

All stocks of Virus III were lost some time before the invention of electron microscopy. Nobody can now be sure what exactly this rabbit-specific virus was, but it was probably Leporid herpesvirus 2, as described by Nesburn in 1969. For an objective summary of the Virus III story, read the page on LHV-2 (p. 380) in The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents (Academic Press, 2012).

“Herpes virus” (Herpes simplex) activity is measured differently. They inject HSV into rabbit brains, and they look for dead rabbits. It seems like this assay should actually be quantitative, based on looking at how long it takes the rabbits to die, but all their rabbits either died within a week or lived longer than a month, so the results were clear without measuring time to death.

 * * *

The results are pretty simple, which is why I would like a summary figure instead of a series of tiny individual tables for each thing they studied.

rivers-summary

Some comments on the data:

  • “Locke’s solution” is like what we call Ringer’s solution, the intravenous fluid given to people who have suffered blood loss. This page indicates that it’s the same as Ringer’s solution, but buffered by bicarbonate instead of lactate. But most other sources indicate that it also includes glucose, making it closer to Tyrode’s solution.
  • Locke’s solution is basically a physiological salt solution, so what’s the difference between that and the “physiological salt solution” used in the phage experiments? The latter doesn’t contain glucose, and isn’t buffered; instead of a pH of roughly 7.6, the pH is “between 6 and 7”.
  • Vaccinia virus was the most stable, retaining the ability to create a skin lesion even after 34 freeze-thaws. Virus III was destroyed after 12, and the other herpesvirus was destroyed after 24.
  • Both bacteria and bacteriophage had more than 99% of their activity destroyed by freeze-thawing in Locke’s solution. All three big viruses were more hardy than that.
  • I don’t know what a “1:10 dilution of trypsin” is. How many USP Trypsin Units is that? What’s the molarity? We have to take Dr. Northrop’s word for it, that it’s the same system for trypsin dilution he always uses.
  • At a mere 1:10 dilution, complement and trypsin were not damaged by 12 freeze-thaws. But when diluted further, they were very susceptible to freeze-thaw.
  • In fact, bacteria and bacteriophage were also more susceptible to freeze-thaw at low dilutions. Even vaccinia virus lost all its effect after 34 dilutions at 1:100,000 dilution, although it didn’t have much activity at that dilution to begin with.
  • Of further interest: the difference between high and low dilutions was only observed in Locke’s solution or salt solution, not broth. Remember these cutting-edge findings when you make your own aliquots.

Data Update: Pox in rabbits, pox in mice

Here’s a paper by Frank Fenner (1914-2010), which compares quite a lot of different poxviruses to establish basic facts about their biology.

By 1958, when this was published, Fenner was already well known in Australia for his leadership role in releasing extremely deadly myxoma virus among the rabbit population. This was deemed to be worth the risk, as the creatures had for almost a century been locust-like in their consumption of Australia’s crops, only making themselves useful as a source of food during the Depression. In a prelude to fellow Australian Barry Marshall’s auto-experimentation with Helicobacter pylori, Fenner and two other experts infected themselves with myxoma to show that the risk to humanity was negligible.

Fenner F (1958). The biological characters of several strains of vaccinia, cowpox and rabbitpox virus. Virology 5(3):502-529. (abstract here: subscription required for full article)

This paper was an attempt to clear up the categorization of various poxvirus strains, mostly vaccinia virus. Fenner got colleagues around the world to provide samples of Mill Hill (V-MH), Williamsport (V-WILL), Pasteur Institute (V-PI), and other vaccinia isolates, which had been designated as either “dermal” or “neuro-” vaccinia based on whether they had been propagated in rabbit skin or rabbit brain. Were these categories practically useful? What strains of vaccinia, if any, were really typical of the virus?

The vaccinia strains were compared to the “Amsterdam” and “Brighton” variants of cowpox, and the “Utrecht” and “Rockefeller Institute” variants of rabbitpox. Rabbitpox is not the same as myxoma virus, by the way.

* * *

Frank Fenner inoculating eggs with virus in 1958. Source: Sydney Medical School

Frank Fenner infecting eggs. Source: Sydney Medical School

Also included in the comparison were “white variants” of certain virus strains. At this time culturing cell lines in vitro was not convenient. Lots of procedures, including growing virus, that we would now do with cellular monolayers in petri dishes were done in a system called the chorioallantoic membrane (CAM). The CAM is the membrane under the shell of an egg. Before doing any experiments on the various viruses, Fenner and associates made sure each virus was pure, by inoculating eggs and then extracting a single “pock” from the CAM for further propagation.

Often they would see that a virus that normally produced red, angry pocks had a few white pocks. Under further study, these “white variants” generally turned out to be milder than their parent strains, as a result of some genetic deletion. The white variants gave me some trouble in putting together the graphs based on Fenner’s data, because they are grouped with non-white strains that are far more virulent.

Sample chorioallantoic membrane pocks. 16a is the "white variant" of 16 (Rabbitpox Utrecht). 15a is the "white variant" of 15 (Cowpox Brighton).

Sample chorioallantoic membrane pocks. 16a is the “white variant” of 16 (Rabbitpox Utrecht). 15a is the “white variant” of 15 (Cowpox Brighton).

Let’s get to the data. After doing a bunch of CAM experiments, Fenner started infecting animals. He compared the viruses for their ability to kill mice and rabbits after brain infection, and induce skin lesions in rabbits after skin infection.

This table contains an immense amount of data, but it takes up two pages which is never a good thing. I think we can turn it into two good figures, one for mouse infection and one for rabbit infection.

fenner-table5

First of all, not all this data needs to be graphed. If you look at the last two columns, every rabbit skin lesion of more than 13 mm in diameter is considered “IPC”, and every smaller lesion is “N”. That’s all we need to know. Also, I don’t think it makes sense to list a mean survival time for experiments where almost every mouse survived. It’s a little sketchy to say that mice that never came close to dying had a “survival time” of 14 days, and then average that arbitrary number with actual survival times of mice that died. So I’ll drop the “mean survival time” data as well.

* * *

My mouse figure has two parts. I didn’t do any statistics on the data.

1A shows the relative virulence, which is a somewhat confusing metric that compares the LD50 (dose of virus at which half of infected animals die) with the virus’s ability to cause pocks on the egg membrane. All of the viruses grow pretty well in eggs, so this shows which viruses are particularly adapted to be dangerous to mice (or rabbits).

1B shows how many mice survived brain infection with each virus strain. Nowadays we would show survival curves for this – you know, the lines that always start out horizontal with 100% survival, and as the experiment goes on, the line goes down and down, stepwise, as mice succumb to mortality. But Fenner just shows the percent of mice that died during infection (probably this means the number that were dead after 14 days). I changed this to become a graph that shows the % of mice that survived infection.

Infect mice. (A)

Figure 1. Mice are more susceptible to neurovaccinia and rabbitpox than dermal vaccinia. (A) For each strain of virus, mice were infected intracranially with a range of doses, and the LD50 was calculated as the dose which was lethal to 50% of infected mice. LD50 values are normalized to the number of CAM pocks produced by the same dose of virus in eggs. Horizontal line represents the limit of detection. (B) For each strain of virus, mice were infected intracranially with 100,000 infectious particles. Here we show the percent of mice surviving to 14 days post-infection (sample size = 10). Bars with white stars represent “white variants”.

The rabbit figure has three parts.

2A is the relative virulence data again. This time it’s not the LD50, but the ID50. (Even though it says “LD50” in Fenner’s table, it says “ID50” in the legend and in the text.) “LD” means lethal dose, whereas “ID” means infectious dose. So this is the dose of virus at which half of infected animals show signs of infection. “Infection” in this case would be a skin lesion. He doesn’t say exactly how big a lesion needs to be before it’s considered “infection”.

2B is a similar graph showing the diameter of the lesions, in millimeters.

And then there’s a bunch of survival curves. 2D is too busy to be able to decipher each datapoint, with eight overlapping lines in a single graph, but what’s important is that there’s a huge difference between 2C and 2D.

rabbit

Figure 2. Rabbits are more susceptible to neurovaccinia and rabbitpox than dermal vaccinia. (A) For each strain of virus, rabbits were infected intradermally with a range of doses, and the ID50 was calculated as the dose which produced a lesion in 50% of infected mice. ID50 values are normalized as described in 1A. (B) For each strain of virus, rabbits were infected intradermally with 100,000 infectious particles. After 5 days of infection, the diameter of each skin lesion was measured with a ruler. (C-F) For each strain of virus, rabbits were infected intradermally with 100,000 infectious particles, and survival was monitored daily (sample size = 2-4).

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