DE2264028A1 - METHOD OF BREEDING A MUSHROOM INFECTED WITH RIBONUCLEIC ACID (DOUBLE STRANDED) VIRUS - Google Patents

Description

"Method for Growing a Ribonueleic Acid (Double-Stranded) Virus infected fungus "

Priority: January 5, 1972, Gi-oss Britain, No. 375/72

The invention relates to a method for growing on an industrial scale Scale of ribonucleic acid viruses in guest fungi.

It is known per se that double-stranded ribonucleic acids and their salts, hereinafter abbreviated as ds-RNA, are effective Represent induction agents for interferons and therefore of particular importance for the broad spectrum prophylaxis of viral infections and are, to a lesser extent, treatments for such infections. It was found to be both natural occurring as well as synthetic double-stranded ribonucleic acids possess such interferon-inducing activity. In particular, such interferon-inducing became natural ds RNA types found in the form of virus particles that are found in some

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Strains of Penicillium chrysogenum, Penicillium funiculonum and Penicillium stoloniferum occur. These ds-RiJS viruses were also found in some other fungal species, e.g. in some strains from Aspergillus niger and Aspergillus foetidus.

To such virus-infected fungi for the subsequent isolation To breed from ds-RNA virus, it was previously necessary to find naturally occurring fungi that were already infected with viruses. The yield of RNA virus particles that is obtained after such cultivation then depends on the specific Combination of 'guest fungus and virus species found by chance.

If, on the other hand, a virus could be found to infect a previously virus-free species of fungus with a selected virus, then one could freely choose the fungus strain that delivers the best results, for example the highest yields or the purest preparations. In addition, it would then be possible to select a specific type of RNA virus as the preferred induction agent for interferon formation in advance. Taking this special type of virus into account, it would then also be possible to infect the most suitable guest fungus with it and to obtain such high yields of the desired virus without the need for a long previous search for a fungus strain which is very uncertain with regard to the outcome. P. Lhoas has described in Nature, 2; 5O (1971) page 248, a method with which he infizier0 a previously uninfected fungus through hyphae anastomosis. However, this method is limited and is for breeding purposes

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not suitable on the industrial scale of virus particles. In Belgian patent 78O 226 there is a better method described in which the cell bodies of the host fungus are infected and grown into infected fungal colonies.

It was the object of the invention to provide a method that can be carried out on an industrial scale in order to remove an uninfected fungal strain to infect with ds-RNA virus particles.

The subject matter of the invention is thus a method for cultivating an infected with ribonucleic acid (double-stranded) Fungus, which is characterized by the fact that one has fungal cells of different sexes or female and male gametes in contact with ribonueleic acid virus particles obtained from mushrooms brings, at least some of the resulting zygotes infected with virus particles and the infected zygotes to one infected fungus.

In the method according to the invention, it is true that two fungal cells of different sex, or two morphologically differentiated or undifferentiated yarns, with the virus particles bring into contact, but preferably contact is made with the fungal cells of a living colony, the cells of different types Sexual or differentiated or indifferentiated gametes contains, with virus particles. If this more suitable method is used, one first establishes a growing colony of the desired one Host fungus is produced by placing the fungus cells of different sexes in two separate colonies or on a suitable one Culture medium mixes. But you can also get a wild one

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or mutant fungus that already has cells of different sexes contains, use.

The cells of the growing colony are then infected with the virus particles brought into contact. In general, the liquid cooling medium containing the growing fungal cells is added a suspension of the virus particles in the same culture medium or in another liquid medium that supports the growth of the Fungi, or at least the pairing, does not interfere, e.g. phosphate buffer containing distilled water (pH about 7). To achieve a good infection, one preferably uses a very dense virus particle suspension. The mixture of fungal cells and virus particles in the culture medium is then used for some Incubated minutes to a few days to clear the infection at least of some zygotes that arise from the pairing of cells or gametes. In general it is sufficient incubate the mixture of fungal cells and virus particles for a few hours at room temperature. At the end of the infection period the infected zygotes are grown.

After the infection, the fungal cells, which of course also include the infected zygotes, are centrifuged and distilled V / ater suspended again. The centrifugation and suspension can be repeated to remove the free virus particles remove the cells. It is very unlikely that they all resulted from mating in the presence of virus particles Zygotes are infected. Therefore, the fungal cells separated after infection contain uninfected, unpaired cells

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or gametes, uninfected and infected zygotes. This Cell mixture is relatively unsuitable for further large-scale cultivation of virus particles. Therefore separates after infection, the infected zygotes are removed from the cell mixture and grow colonies from these infected zygotes. The difficulty now is to get the infected zygotes out isolate the cell mixture.

For example, one can have samples of the cell mixture in one Cultivate the medium in the form of plate cultures, which can be converted into cell colonies grows. These cell colonies are then im Electron microscope examines the infected colonies, i.e. those that have emerged from infected zygotes locate. However, this method is cumbersome.

The dye methylene blue stains dead Pils-Zellon, but not living fungal cells. If colonies of infected zygotes grow in a culture medium that contains methylene blue, this is shown some of the colonies have a central zone with dead cells that are stained blue by the dye. This observation leads in some special cases a suitable method of identifying the infected zygotes.

In some cases, the fungal cells can be of different sexes fail to produce the essential nutrient. After mating, however, the resulting zygotes and their offspring can produce this essential nutrient. That is, such a fungus is infected in the method according to the invention and the resulting cell mixture is in the culture medium to which the

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lacking essential nutrients, cultivated in the form of plate cultures, so only zygotes and their offspring can survive. However, if the medium contains methylene blue, some of the colonies have derived from infected zygotes, a central zone of dead cells stained blue by the dye likely killed by the virus. One can do plate 'cult doors make these "blue" colonies from a fresh culture medium, the colonies produced in this way then have one very high virus particle concentration. This procedure can as often as necessary, to be repeated and allows selection a blue colony that after breeding and overculturing contains a steadily growing proportion of blue colonies on plates. Serve colony. Is replicated and serves as Stem colony for the large-scale breeding of infected seeds.

Although this method can only be used for special cases, the electron microscope according to the invention can be used accordingly Procedure to locate infected colonies from infected zygotes. This way you can stock crops of colonies which after repeated subculture in the Examination in the electron microscope constantly high concentrations of virus particles to have.

Correspondingly, the concentration of virus particles produced by a colony can be examined by gel electrophoresis and in this way select the best colonies for the stock cultures.

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The method according to the invention can also be applied to a fungus apply, which see at least partially by fusion of cells of different sexes or by fusion of gametes increased.

The yeasts are an example of mushrooms of the Ascomyeetes tribe, which multiply at least in part by fusing cells of different sexes. But at v / eitem the biggest Part of the mushrooms classified as Ascomyeetes partially reproduces by fusion of morphologically differentiated or undifferentiated Gametes. Also the mushrooms Saprolegniales, which belong to the Phycomycetes, reproduce like the earth mushrooms Mucorales and Peronosporales, partly by fusion of the Gametes.

By means of the method according to the invention it is possible to obtain a previously uninfected fungal strain with ribonucleic acid virus particles to infect at will. Appropriate selection of the guest mushroom is therefore also available on an industrial scale maximum yields of virus particles. In addition, fungal virus particles, containing a particularly preferred type of double-stranded ribonucleic acid in any selected one Guest fungus are cultivated, regardless of whether this guest fungus also shows such a virus infection in nature or not. For example, virus particles isolated from a fungus of the strain Penicillium or Aspergillus in a Yeast are grown.

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The examples illustrate the invention.

example 1

Infection of fungal cells of different sexes of the strain Saccharomyces cerevisiae with ribonucleic acid (double-stranded) virus from Aspergillus niger and Penicillium funiculosum.

Ribonucleic acid (double-stranded) virus from an Aspergillus strain niger IMI 14689-1 and from a strain Penicillium funicu3osum IMI I63167 is in V / water, the phosphate buffer, pH 7 * 2,

contains, suspended. The suspension contains about 10 virus particles per ml. Two adenine-requiring strains of Saccharoniyees cerevisiae are used: ad-lo6 and ad-2.1a (described by DR Woods and EA Bevan in J. gen. Microbiol., JU (1968) page 115 and in JM Somers and Bevan EA. in Genet Res. Camb., \] j_ (I969) page 71). All media are described in this last citation from the literature. The incubation is carried out ^{at 28 ° C.} For the infection, a loop full of fungal cells of each sex is mixed on a complete agar medium, pH 5> 8, and 0.01 ml of the virus suspension (10 ″ particles / ml) is added Incubated for hours, then the cells are collected, washed by centrifugation twice at 800 g and resuspended in distilled water. To isolate the zygotes or the unpaired cells or the zygotes alone, since the ad cells are complementary, are Complete medium or minimum medium, pH 5.8, plate cultures prepared.

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Incubation shows 1 to 5 percent of the zygotes that have arisen Colonies have a central dark blue zone of dead cells. These colonies are referred to as "blue colonies", im Contrasted with the normal white diploid colonies. From five of these blue colonies are on a complete methylene blue medium Plate cultures made. Blue colonies always develop, but their proportion varies between 10 and 70 percent, The single cells are isolated three times in succession from these blue colonies. In any plate culture there are more than 95 percent of the colonies are blue. The virus suspensions are prepared from three blue colonies from each plate culture and by Examined gel electrophoresis and electron microscope. They all contain the virus, while 20 examined white diploids Colonies are virus-free. One of these infected diploid colonies is grown on complete agar medium, pH 5.8. Then thin sections are made for electron microscopy. There are many virus particles in the cytoplasm of the cells,

Example2

Infection of fungal cells of different sexes from Saccharomyces cerevisiae with ribonucleic acid (double-stranded) -Vlrus from Penicillium chrysogenum and Penicillium stoloniferum.

According to Example 1, virus suspensions of Penicillium stoloniferum ATCC 14586 and from Penicillium chrysogenum ATCC 10111 and two strains of Saccharomyces cerevisiae, ad, - ^ and the wild type a is used. The infection is carried out according to Example 1, but with the difference that both

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the plate cultures on methylene blue minimal medium of the white haploid V / ildtyps a and the white diploid colonies and isolate the blue diploid colonies. The colonies that develop from the zygotes are somewhat larger than the colonies that develop and can develop from the haploid cells can be distinguished with a little experience. After the 14-day Incubation is 1 to 5 percent of that developing from the zygotes Colonies "blue" colonies. Plate cultures are prepared from 5 blue colonies on methylene blue complete medium. According to Example 1, the individual cells are isolated three times in a row. Again, the virus is just In the blue colonies.

The number of wild-type a haploid colonies on the methylone blue minimal medium dilution plates cultured v / ground, e.g. after making the plate culture of the resuspended Cells after mating can optionally be reduced by using a substantial excess, e.g. tenfold excess, of the marked (lacking food) haploid strain. In this way, the wild type becomes in the Serial dilutions on the methylene blue minimal margin plates retired earlier.

meeting

It is believed that mating is necessary for infection. This assumption is confirmed by two observations; Point to methylene blue complete medium in the PJattenkulturen the unpaired parent cells the central zone of dead

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Also, cells that are observed in haploid colonies after mating an infected diploid cell will not be 5 zygotes mating in the absence of virus particles emerged in the presence of virus particles and under the conditions under which the mating fungal cells infected have been bred. There is no blue colony. 5 colonies of each zygote are formed on the 3 edge of the plate cultures selected to be checked for virus. They are virus free.

The results already available give some clues as to how the infection of mating fungal cells occurs, the low percentage of infected zygotes and eggs changing proportion itself from the newly infected zygotes forming infected Cells indicate that only a few virus particles penetrate the mating fungal cells, possibly when part of the cell wall to which they are adsorbed is destroyed during mating. Some cells that are right after have opened during the pairing, do not take up any virus particles. In each infected cell, however, the number eventually reaches on virus particles the height that causes the death of the cell.

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Claims (1)

Claims 1. Method of growing a ribonucleic acid (double stranded) virus infected fungus, thereby characterized by having fungal cells of different sexes or female and male gametes with fungal ribonucleic acid virus particles, at least some of the resulting particles Zygotes are infected with virus particles and the infected zygotes are grown into an infected fungus. 2. The method according to claim 1, characterized in that after contacting the fungal cells with the virus particles cultivated many separate fungal colonies and each derived from an infected zygote with virus particles continues to grow infected colony (s) to the desired amount of infected fungus. 5. The method according to claim 1 and 2, characterized in that that virus particles from infected fungi of the strains Penicillium or Aspergillus, in particular Penicillium chrysogenum, Penicillium stoloniferum, Penicillium funiculosum, Aspergillus niger or Aspergillus foetidus. H. Verfixhren according to claim 1 to ~ 3, characterized in that yeast cells of different sexes are used as fungal cells.