Rabbit mosaic virus?

by Mike

This paper (1) seemed weird, at first. With a title like “Immunologic Reactions with Tobacco Mosaic Virus“, and methods that included injecting the virus into rabbits, I wondered if it was an attempt to use all possible means to infect animals with the legendary plant virus. But no, the author of today’s paper points out in her introduction that that work was already done (2) by Maurice Mulvania of the Tennessee Experiment Station, and we now know (as of 1929) that the virus doesn’t reproduce in rabbits.

So what is she doing, looking at the rabbit’s immunologic reactions to tobacco mosaic virus (TMV)?

Well, primarily, trying to back up the evidence that the virus exists at all, as a physical object. The introduction to this paper is particularly interesting in its snapshot of what people thought about viruses, before electron microscopy.

The properties of tobacco virus are frequently linked to those of enzymes. An apparent similarity between them may be due to the fact that they have both been studied in tissue extracts and in an impure state.

In Angela Creager’s 2001 book The Life of a Virus: Tobacco Mosaic Virus as an Experimental Model, 1930-1965, a similar point is made:

Investigations of virus infectivity resembled contemporary biochemical studies of enzyme activity under various conditions. Allard, mentioned above for his insistence on the contagious nature of TMV, assessed the effects of heat, germicidal agents, and chemical reagents on viral infectivity… A few years later, Maurice Mulvania conducted similar studies assessing the effects of light rays, heat, dialysis, and exposure to bacteria on the infectivity of TMV, publishing his work in the other major journal for research on plant diseases, Phytopathology. The susceptibility of TMV to some of these inactivating agents led him to speculate that the virus might be a simple protein or enzyme.

So the word “virus” did not mean “virus” as we now know it, an organism with genetic material that can reproduce when it infects host cells. A “virus” could simply be a toxic substance. After all, “virus” comes from the Latin word for poison.

Helen Purdy, later Helen Purdy Beale, was a legendary figure in plant virology. Here is a 2006 review of her work (3) that mentions, right off the bat, this 1929 paper in which she immunized rabbits against TMV-infected plant saps and uninfected plant saps, and concluded that the virus saps had unique proteins which were not present in the normal plants. In the introduction she also gives a mini-literature review of what people thought TMV might be.

purdy-1929-introduction

Verdict of history:

Light and electron microscopy of cauliflower mosaic virus in Brassica rapa cells, from Martelli and Castellano, 1971 (5). "IB" means inclusion bodies.

Light and electron microscopy of cauliflower mosaic virus in cells, from Martelli and Castellano, 1971 (5). “Ib” means inclusion bodies. Click for higher resolution.

  • Egiz: Wrong!
  • Hunger: Wrong! That sounds like a prion disease.
  • Beijerinck: Right about the “contagium vivum” part, wrong about “fluidum”. Beijerinck is generally credited as the first scientist to demonstrate that an infection could be transmitted by something smaller than a bacterium.
  • Heintzel/Woods/Chapman: Wrong!
  • Iwanowski: Right about being unculturable, wrong about being bacterial.
  • Allard: Right!
  • Palm: Right! Those inclusions are made up of solid particles of the infectious virus.
  • Olitsky: Wrong!

Even Palm didn’t contemplate a new form of “parasite” other than extra-tiny bacteria. His paper (4) was written in Dutch and published in the now-elusive Bull. Deliproefsta. te Medan-Sumatra. This publication is so elusive that I can’t figure out what “Deliproefsta” stands for. Among others interested in Palm’s findings, Dr. L.O. Kunkel provided a synopsis at the end of an article in the 1921 Bulletin of the Experiment Station of the Hawaiian Sugar Planters’ Association. (This is what came up first from a web search for “tobacco” and “deliproefsta”.

In a recent paper Palm has described certain “amoebiform corpuscles” which he finds in mosaic tobacco leaves. He apparently considers these bodies to be analogous to those of corn mosaic. In addition to the “larger foreign corpuscles,” which he says “lie either in intimate contact with the nucleus or more or less in its vicinity”, he finds numerous extraordinarily small granules which, he thinks, may represent an orgnaism. On the assumption that the granules are alive and that they are the same bodies which Iwanowski described as bacteria, he gives them the name Strongloplasma iwanowskii.

In considering the intracellular bodies as a possible cause of mosaic disease, the writer meets the same difficulty which prevented Iwanowski from looking on the “Plasmaanhaufungen” as causal organisms. They are too large to pass through bacterial filters. Iwanowski did not know, however, that these bodies are associated with mosiac disease in several different plants. Perhaps this further evidence may justify an attempt to harmonize statements which at first appear to be contradictory. It may be that the amoeboid bodies… represent only one stage in the life of a causal organism. At another stage they may be so small and plastic that they can pass through the fine pores of a filter and escape detection under the microscope. They probably become visible only after a certain period of growth within the host cell.

Bingo!

* * *

The 1929 Purdy paper has a remarkably coherent set of conclusions that agree with what we now know. Purdy did a large number of experiments that mostly involved immunizing rabbits with “virus-saps” and “normal saps” from tobacco, tomato, pepper and petunia plants. She then took the immune sera from these rabbits and saw if they could block the activity of the various saps. There’s a whole page of “summary” and conclusions. In brief:

  • Tobacco virus-saps contain all the antigens that normal tobacco saps contain, plus a few others.
  • Virus-saps from tobacco, tomato, pepper and petunia plants all have some antigens in common, which are probably not found in normal sap.
  • Normal rabbit or guinea pig serum cannot block TMV from infecting plants. Neither can serum from animals immunized with normal plant sap. These sera do not contain the proper antibodies. But serum from animals immunized with virus-saps can block TMV from causing plant disease.

Well, that’s pretty conclusive. But these plant saps are mostly just soluble proteins. So we still don’t know if the virus is made of of particles, or if it is liquid in nature. Some sort of extra-sensitive microscope would be very helpful.

* * *

1. Purdy HA (1929). Immunologic reactions with tobacco mosaic virus. J Exp Med 49(6): 919-935.

2. Mulvania M (1926). Studies on the nature of the virus of tobacco mosaic. Phytopathology XVI: 853-872.
(note: I can’t find any archive that includes Phytopathology any earlier than 1970, which is a shame since a lot of papers and indices refer to Mulvania’s work).

3. Scholthof KBG, Peterson PD (2006). The role of Helen Purdy Beale in the early development of plant serology and virology. Adv Applied Microbiol 59: 221-241.

4. Palm BT (1922). Die Mosaikziekte vac de Tabakeen chlamydozoonose? Bull Deliproefsta te Medan-Sumatra xv: 7-10.

5. Martinelli GT, Castellano MA (1971). Light and electron microscopy of the intracellular inclusions of cauliflower mosaic virus. J Gen Virol 13: 133-140.

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