JPH1198979A - Virus capable of carrying out proliferation and lysing algae by specifically infecting red tide plankton and control of red tide utilizing the same virus and red tide controlling agent and storage of the same virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain virus targeting algae of the genus Heterosigma which is a plankton which causes red tide and provide a method for controlling red tide using the virus and provide a method for storing the virus. SOLUTION: This virus specifically infects algae of the genus Heterosigma and can proliferate. This method for isolating the virus comprises steps concentrating a liquid sample containing algae of the genus Heterosigma, culturing the resultant concentrate, filtering the resultant culture medium by a filter, inoculating the resultant filtrate into the culture medium of algae of the genus Heterosigma to culture the virus, subjecting the culture medium in which lysis of algae of the genus Heterosigma is observed to limiting dilution with culture medium of algae of the genus Heterosigma and cloning the virus. This red tide controlling agent contains the virus as an active ingredient. This method for controlling red tide comprises spraying the virus into red tide area. This method for storing the virus comprises freezing the liquid containing the virus in the presence of dimethyl sulfoxide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a virus that can specifically infect and multiply red tide plankton, a red tide control method and a red tide control agent using the virus, and a method for preserving the virus. The present invention relates to a virus capable of specifically infecting and multiplying an algae of the genus Heterosigma, a red tide controlling method and a red tide controlling agent using the virus, and a method for preserving the virus.

[0002]

2. Description of the Related Art The marine aquaculture industry in Japan accounts for about 1/4 of the total domestic fishery production value. In this promotion, it is indispensable to protect the environment of the aquaculture and fishing grounds in particular, and it is extremely important to promote effective countermeasures against the red tide that causes serious damage. Against this background, various measures to control red tide damage have been proposed. However, these are emergency evacuation measures such as spraying clay into red tide waters (submersing plankton physically on porous clay and submerging it) or spraying chemicals such as hydrogen peroxide (chemically killing plankton). However, there were problems in terms of scale and cost, and the impact on the ecosystem due to the introduction of artificial foreign substances into natural waters, that is, safety was a concern. . Therefore, in the sea area where the red tide actually occurred,
The current situation is to stop feeding farmed fish and evacuate fish cages to clear waters to surpass the emergency.
Therefore, there is a demand for the development of a red tide control technology which is practical in terms of scale, cost and safety.

Until very recently, the fishery damage caused by heterosigma acacio, the target organism of this study, was relatively mild due to the extremely large amount of fishery damage caused by other red tide-causing species (genus Shutnera, Gymnodinium, etc.). It has been thought that there is. However, in recent years, the amount of damage per red tide caused by this species has been increasing, and since 1995, about 50% of the major red tide damage in Japan has been caused by heterosigma, and the future behavior of this plankton has attracted attention. ing. Overseas, the threat of the heterosigma red tide to the aquaculture industry is being recognized internationally, including over 1 billion yen in damage to aquaculture salmon in Big Glory Bay in New Zealand.

[0004]

SUMMARY OF THE INVENTION Accordingly, the present invention provides a method for controlling red tide and a red tide controlling agent using a virus, which targets heterosigma, which is a plankton causing red tide, which is of great industrial interest. The purpose is to: Another object of the present invention is to provide a virus targeting Heterosigma and a method for isolating the virus.

Another object of the present invention is to provide a method for preserving a virus targeting Heterosigma. Another object of the present invention is to provide a method for reproducing a virus stored by the above method.

[0006]

Means for Solving the Problems The present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, have succeeded in isolating a virus capable of specifically infecting and multiplying heterosigma acacio. The invention has been completed. The present inventors have also found a method for stably storing a heterosigma acaciovirus in liquid nitrogen in the presence of dimethyl sulfoxide and a method for regenerating a heterosigma acaciovirus stored by this method.

[0007] That is, the present invention provides a virus capable of specifically infecting and multiplying an algae of the genus Heterosigma.
The algae of the genus Heterosigma may be Heterosigma acacio. A virus that can specifically infect and multiply heterosigma akashio lacks tail and outer membrane structures and has a particle size
It may be a virus having a icosahedron of 202 ± 6 nm and having double-stranded DNA. Further, the present invention provides a method of filtering a liquid sample containing a heterogeneous algae that is infected with a virus capable of specifically infecting and multiplying a heterogeneous algae with a filter having a pore size of 0.2 μm, and obtaining the obtained filtrate. Is inoculated into a culture of heterosigma algae, cultured, and the virus is cloned by limiting dilution of the culture in which the lysates of heterosigma algae were observed with the culture of heterosigma algae. And a method of isolating a virus capable of specifically infecting and multiplying an algae of the genus Heterosigma. This isolation method may further include a step of irradiating the liquid sample with ultraviolet rays. Irradiation with ultraviolet light may be performed at any time during the virus isolation operation, for example, before or after filtration of the liquid sample. Furthermore, the present invention provides a red tide controlling agent comprising, as an active ingredient, a virus capable of specifically infecting and multiplying heterosigma algae. Furthermore, the present invention provides a method for controlling red tide, which comprises spraying a virus capable of specifically infecting and growing on algae of the genus Heterosigma in red tide waters.

[0008] The present invention also relates to a method for preserving a virus capable of specifically infecting and multiplying an algae of the genus Heterosigma, wherein a liquid containing the virus is frozen in the presence of dimethyl sulfoxide. The above method is provided. In a preferred embodiment of the present invention, a liquid containing a virus capable of specifically infecting and multiplying heterosigma algae is frozen in liquid nitrogen in the presence of dimethyl sulfoxide at a concentration of 10 to 20% by volume. . According to the method of the present invention, a suspension of a virus capable of specifically infecting and multiplying heterosigma algae was prepared and inoculated into a fresh culture solution of heterosigma algae, and lysed alga was confirmed. At this point, the method may include a step of mixing the culture supernatant with the dimethyl sulfoxide-containing medium and freezing the mixture in liquid nitrogen. As a specific example, a fresh culture solution of heterosigma algae is inoculated with a 1/100 to 1/50% by volume of the virus suspension, and when lysed alga is confirmed, the culture supernatant is And a SWM3 medium containing dimethylsulfoxide at a concentration of 20 to 40% by volume are mixed in equal amounts, and the mixture may be frozen in liquid nitrogen at -196 ° C.

[0009] The present invention also provides a method for regenerating a virus that can be specifically infected to algae of the genus Heterosigma and proliferates, which is preserved by the above method, wherein the frozen solution containing the virus is thawed. And inoculated into a fresh culture of heterosigma algae. In this regeneration method, it is preferable that a fresh culture solution of algae of the genus Heterosigma is inoculated with a thawed virus-containing solution in an amount of 1% by volume or less. Virus stored and regenerated by the method of the present invention,
It retains infectivity against algae of the genus Heterosigma.

[0010] In the present specification, "red tide" refers to a phenomenon in which the color of a water area changes with the rapid proliferation of plankton. This term is widely used with or without aquatic product damage. Typical examples of plankton that cause harmful red tide in Japan include Schutnella, Gymnodinium, and Heterosigma. The term "algae of the genus Heterosigma" belongs to the class Raffid alga, and heterosigma akashio is known as a species belonging to the genus Heterosigma.

[0011] " Heterosigma ak
ashiwo ) ”is a flat ellipsoid with a major axis of 11 to 25 μm.
It is a unicellular marine phytoplankton with book flagella. Heterosigma acacio grows in asexual reproduction by dichotomy. Although the sexual reproduction process has not yet been elucidated, it is known that it forms cysts (cells corresponding to a kind of seeds) in winter and is dormant in submarine mud. Heterosigma akashio is known as a causative species of harmful red tide that occurs in the temperate or subarctic regions of the north and south hemispheres. In Japan, red tides form in coastal areas from May to July, often causing damage to cultured fish. Although the mechanism of killing fish by heterosigma acacio has not been fully elucidated, it has been pointed out that it may be due to abnormal secretion of gills or the effect of hemolytic substances.

[0012] Further, the term "virus" refers to an infectious spherical or fibrous microstructure capable of growing only in an infected host cell. Viruses are completely different from bacteria in that they cannot multiply and do so only by utilizing the biosynthetic system of the host cell. The feature is that the host specificity is significantly higher than that of bacteria. The term "host specificity" is a term indicating the extent to which the range of types of host organisms that can be infected and propagated by a pathogenic parasite (virus in this case) is limited. For example, the phrase “high host specificity” or “narrow host range” means that a small number of organisms can be infected.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
Heterosigma akashio, the target organism of this study, is a kind of harmful red tide plankton distributed in inland areas such as the Seto Inland Sea. This species has been attracting attention for a long time as a plankton species that causes fisheries damage, and in particular, tends to cause large-scale fishery damage in recent years.

The virus of the present invention is capable of infecting and multiplying algae of the genus Heterosigma. A virus (HaV: heterosigma acaciovirus) that can specifically infect and multiply heterosigma akashio is a icosahedron having a tail structure and an outer membrane structure, a particle diameter of 202 ± 6 nm, and a double-stranded structure. Has DNA. In experiments, heterosigma acacio cells infected with the virus lost motility and died, releasing a large number of replicated viruses,
It induced infection and death of new (ie, uninfected) heterosigma acacio cells. Therefore, it is possible to completely kill all cells in culture within at least about 10 days by adding only one virus to a culture solution containing millions to tens of millions of heterosigma acacio cells. It is possible. In addition, the host has high host specificity, and to date no effect of the virus on phytoplankton other than heterosigma acacio has been detected.

The virus of the present invention can be isolated as follows. First, a liquid sample, such as seawater, containing an algae of the genus Heterosigma, which is infected with a virus that can specifically infect and grow on algae of the genus Heterosigma, is prepared and stored at a temperature of about 4 ° C. . As the algae of the genus Heterosigma, heterosigma akashio is preferable.
The liquid sample may be irradiated with 15 W of ultraviolet light for 0 to 120 seconds. This operation is effective in inducing the algicidal activity of the virus. After the obtained solution is filtered through a filter having a pore size of 0.2 μm, the filtrate is inoculated into a culture of an algae of the genus Heterosigma.
^-2 s ^-1 , light for 14 hours: Incubate for 1 to 2 days under dark conditions for 10 hours. Heterosigma algae culture was prepared by cloning the algae by micropipette method or limiting dilution method in advance and establishing a strain. At 20 ° C., a modified SWM3 medium containing ₂ nM Na ₂ SeO ₃ (Chen et al., 1969, J . Phycol 5: 211-220; I
toh & Imai, 1987, Shuwa, Tokyo, p.122-130), 45 μmol ph for 5-7 days with a 10-hour light / dark cycle
It can be prepared by culturing under the irradiation of white fluorescent light of otons m ^-2 s ^-1 .

Then, the virus is cloned by limiting dilution of the culture in which the lysing algae of the genus Heterosigma is observed with the culture of the algae of the genus Heterosigma. The method for preparing the culture solution of the algae of the genus Heterosigma is as described above. The limiting dilution may be performed as follows. First, the supernatant of the culture solution in which the lysis of heterosigma algae was observed was taken, and serially diluted with the culture solution of heterosigma algae to prepare a 10-fold dilution series of the supernatant. For example, the cells are cultured at 20 ° C., 45 μmol photons m ⁻² s ⁻¹ , 14 hours light: 10 hours dark for 7 to 10 days. After the cultivation, the highest dilution in which the lysing alga of the genus Heterosigma was observed is selected, and the above operation is repeated at least once using the supernatant.

After the final limiting dilution, the supernatant of the highest dilution in which the lysing algae of the genus Heterosigma was observed was inoculated into a large-scale culture (eg, about 25 ml) of the algae of the genus Heterosigma. Culture at 20 ° C., 45 μmol photons m ⁻² s ⁻¹ , 14 hours light: 10 hours dark for 3 to 5 days. The culture solution in which the lysing alga of the genus Heterosigma is observed may be stored in a dark place at 4 ° C. after addition of 0.2% sodium azide. This culture solution was filtered through a filter having a pore size of 0.02 μm to capture virus particles on the filter,
This can be stained with DAPI (4 ', 6'-diamidino-2-phenylindole) and observed under ultraviolet light.

The virus of the present invention can be cultured at relatively low cost (albeit on a small scale) in an laboratory system. The red tide can be controlled by spraying the virus of the present invention on the red tide water area. In controlling the red tide, a culture solution of the virus of the present invention or a supernatant thereof may be used as it is, or a preparation containing the virus of the present invention as an active ingredient may be prepared and used.

The method for controlling red tide and the agent for controlling red tide of the present invention utilize a virus having a high host specificity already existing in the natural environment. Has high expectations.
Further, the red tide controlling agent of the present invention, unlike other ordinary agents, has a self-replicating ability in the virus itself, so that a wide range of red tide control can be expected with a small amount of injection. Next, an example of a specific procedure of a method for storing and reproducing a virus that can specifically infect and multiply heterosigma algae will be described below.

1. A culture solution of heterosigma algae is prepared. For example, as described in Example 1 described below, a culture solution of an algae of the genus Heterosigma may be prepared.

2. A suspension of a virus capable of specifically infecting and multiplying heterosigma algae is prepared. For example,
A virus suspension is prepared as described in Example 2 below.

3. A fresh heterosigma culture is inoculated with the virus suspension. For example, as described in Example 3 below, 2. Is inoculated into a culture of fresh heterosigma algae and cultured. Observe the culture with an optical microscope to check for lysed alga. At this time, spherical infected cells may remain.

4. When lysed cells are confirmed, the culture supernatant is
20-40% by volume dimethyl sulfoxide (hereinafter referred to as "DMS
O ". ) Is rapidly mixed with an equal volume of the SWM3 medium, and the mixture is dispensed into vials.

5. The vial is directly immersed in liquid nitrogen at -196 ° C and frozen. 6. Store vials in liquid nitrogen as is. 7. When necessary, remove the vial and quickly thaw it in running water until a rice-sized ice block remains.

8. The thawed virus-containing solution is inoculated into a fresh culture of heterosigma algae. However, DMS
To prevent the toxicity of O itself from affecting heterosigma cells, the inoculum should be less than 1% by volume of the culture. 8. When the culture is cultured, the virus infects algae of the genus Heterosigma, and lysates are observed. Therefore, it can be seen that the virus stored and regenerated by the above-described method retains infectivity against algae of the genus Heterosigma.
Hereinafter, the present invention will be described specifically with reference to examples. The scope of the present invention is not limited by these examples. In the following examples, unless otherwise specified,%
Denotes the volume%.

[0026]

【Example】

[Example 1] Culture of algae Heterosigma acacio and other microalgae strains used in the examples were cloned by the micropipette method or the limiting dilution method and established. Heterosigma acacio strains are at 20 ° C, other algae strains are at 15 or 20 ° C.
In a modified SWM3 medium containing ₂ nM Na ₂ SeO ₃ (Chen et al., 1969,
J. Phycol 5: 211-220; Itoh & Imai, 1987, Shuwa, Tok
yo, p.122-130) and cultured under a white fluorescent lamp of about 45 μmol photons m ⁻² s ⁻¹ in a light / dark cycle of 14 hours: 10 hours.

[Example 2] Isolation of virus Seawater collected from Nomi Bay, Kochi Prefecture, Japan, on which the heterosigma red tide occurred on July 11, 1996 (contained 35400 heterosigma acacio per ml). ) At 4 ° C and 24
Transported to the laboratory within hours. Processing in the laboratory is Br
atbak et al. ((1996), J. Mar. Syst. 9: 75-81) and Jacob
It is a modification of the method of Sen et al. ((1990), J. Phycol 32: 923-927). Place 50 ml of the collected seawater sample in a Petri dish, irradiate ultraviolet rays (wavelength: 254 nm, Toshiba GK15) for 0, 30, 60, 90 and 120 seconds, respectively, and incubate at 20 ° C for 2 days by the same culture method as in Example 1. did. Thereafter, each sample was filtered through a Nuclepore membrane filter having a pore size of 0.2 μm, and 200 μl of each filtrate was subjected to Heterosigma acacio GS95.
Was inoculated into 5 ml of the culture solution, and cultured under the same conditions as in Example 1. Cultures were checked daily by light microscopy to check for the presence of cell lysates. Lytic algae were detected in heterosigma acacio GS95 inoculated with a seawater sample irradiated with ultraviolet light for 0, 30 and 60 seconds, but were not detected in a seawater sample inoculated with ultraviolet light irradiated for 90 and 120 seconds.

The virus was isolated by two limiting dilution methods (Suttle & Chen, 1993, Mar. Ecol. Prog. Ser.
92: 99-109). The supernatant of each culture solution in which cell lysis was observed was taken, and 150 μl of a cell suspension of heterosigma acacio GS95 was taken.
A 10-fold dilution series of the supernatant was prepared by serial dilution with. Eight dilutions were prepared. After culturing them, the highest dilution was observed in which the lysed alga of heterosigma acacio GS95 was observed, and the above operation was repeated once more using the supernatant. At this time, the risk factor of the presence of two or more viruses was 0.0106 or less. Finally, in the second limiting dilution assay, the supernatant of the highest dilution at which lysed cells of heterosigma akashio GS95 were observed was added to 50 ml of heterosigma akashio GS95.
Was inoculated into a fresh culture and cultured. The culture solution in which lysates of heterosigma acacio GS95 cells were observed was combined with 0.2% sodium azide and stored in a dark place at 4 ° C. This was tentatively named “first virus suspension”. did. All cultures were performed as in Example 1. At the same time, Nishihara et al., Based on the number of wells showing
((1986), Eisei Kagaku 32: 226-228) Using a computer program developed, the concentration of virus in each assay was calculated.

The lysed cells of the cells of Heterosigma akacio GS95 were filtered with a filter having a pore size of 0.02 μm (Anodisc 25, Whatman I).
(nternational Ltd.) to capture the virus particles on the filter, which is then DAPI (4 ', 6'-diamidi
No-2-phenylindole) and observed under a fluorescence microscope. The lysate was observed to contain a number of particles stained with DAPI, indicating that the particles (ie, virus) have double-stranded DNA. The virus isolated in this manner was named heterosigma acaciovirus (HaV) and used in Examples 3 and 4 below. Further, it was observed with a transmission electron microscope that the virus particles were negatively stained with uranyl acetate.

[Example 3] Virus host range "First virus (HaV) suspension" obtained in Example 2
In addition, (1) no treatment, (2) filtration with a 0.2 μm pore size filter (DISMIC-25, Advantec), (3) 0.1 μm pore size
m (Anotop ^™ 25, Anotec), or (4) treatment at 100 ° C for 5 minutes.
μl was inoculated and cultured in 1 ml cultures of the algal strains listed in Table 1 during the logarithmic growth phase. The culture was observed with an optical microscope. Strains that did not lyse after 10 days were considered not hosts for the virus. Table 1 shows the sensitivity of various algae strains to HaV infection.

[0031]

[Table 1]

From the results in Table 1, it can be seen that the infectivity of HaV is strain-specific rather than species-specific, indicating the phenotypic diversity of heterosigma acacio strains in viral infection. .

[Example 4] Action of virus on heterosigma akashio [0033] The stock of the "first virus (HaV) suspension" obtained in Example 2 was (1) untreated, (2) pore size 0.2 Filtration with a 2 μm filter (DISMIC-25, Advantec), (3) pore size
Filtration through a 0.1 μm filter (Anotop ^™ 25, Anotec) or (4) treatment at 100 ° C. for 5 minutes, 50 μm each
l was inoculated into 4 ml cultures (triplicate) of the algal strains listed in Table 1 during logarithmic growth. Fluorometer (Turner Des
igns) was used to monitor heterosigma acacio growth. After lysing, the cells in each culture are analyzed using the Hara and Chihara methods.
((1982), Jap. J. Phycol. 30: 47-56) and observed with a transmission electron microscope.

Culture time and heterosigma (heterosigma akashio GS95 (FIG. 1A) and heterosigma akashio UR94)
(B in FIG. 1)) is shown in FIG. In FIG. 1, the vertical axis represents the relative value of chlorophyll fluorescence in the culture solution,
The horizontal axis represents the culture time (days). ■ indicates (1) no treatment, ● indicates (2) 0.2 μm pore size filter (DISMIC-25, Advante
x: (3) Filtration by a filter having a pore size of 0.1 μm (Anotop ^™ 25, Anotec), Δ: (4) 100 ° C
Heterosigma akashio GS95 or Heterosigma akashio UR94 inoculated with a stock of "first virus (HaV08) suspension" treated for 5 minutes.

(1) No treatment, or (2) a pore diameter of 0.
Lytic algae were generated in heterosigma acacio GS95 and heterosigma acacio UR94 inoculated with a stock of "first virus (HaV) suspension" which had been filtered through a 2 μm filter (DISMIC-25, Advantec). In contrast,
Filter (3) having a pore size of 0.1 μm (Anotop ^™ 25, Anot
The algicidal properties were lost by filtration according to ec) or by treating (4) at 100 ° C for 5 minutes. This indicates that the virus is about 100-200 nm in size and thermally unstable.

Further, no processing of (1) or (2)
"First virus (HaV) suspension" filtered through a 0.2 μm pore size filter (DISMIC-25, Advantec)
When the culture solution in which the stock of inoculum Heterosigma akashio UR94 was lysed was observed with a transmission microscope, a virus was observed. The virus is 202 ± 6 nm in diameter (mean ± standard deviation) and is pentagonal or hexagonal in cross-section, indicating that the virus is icosahedral. The virus also had an asymmetric spherical core with a high electron density different from the capsid and lacked a tail.

In contrast, the pore size of (3) was 0.1 μm.
A culture of heterosigma akashio UR94 inoculated with a stock of the “first virus (HaV) suspension” that has been filtered through a filter (Anotop ^™ 25, Anotec) or treated at 100 ° C for 5 minutes in (4). In, no virus replication was detected. As a stage prior to lysis, the cells of heterosigma acacio spheroidized, lost motility, settled on the bottom surface, and then collapsed. These observations suggest that the infected cells sink to the bottom of the water, as observed with natural red tide pollution. It is noteworthy that the cessation of migration over heterosigma acacio cells at the end of the red tide is associated with a loss of motility caused by viral infection.

[Example 5] Separation of HaV from other red tide seawater The seawater collected on June 24, 1996 in the Buzen Sea, Fukuoka Prefecture, Japan, where the heterosigma red tide occurred, was subjected to Examples 2 to 4 described above.
As a result, a plurality of heterosigma acacioviruses (HaV) having similar physical and chemical properties were obtained. FIG. 2 is a negative staining image of one of the strains. The virus diameter is about 0.2 μm.

Example 6 In this example, the effect of light conditions and temperature conditions on HaV infectivity was examined. 5
C., 10 C., 15 C., 20 C. and 25 C., infectious titers (concentration of virus particles having infectivity = 1 / ml) in incubators set in advance by the limiting dilution method (see Example 2). ) Were stored two by two. One of them is shielded from light by aluminum foil (dark condition), and the other is 14 hours: 10
A white fluorescent lamp of 45 μmol photons m ^-2 s ^-1 was irradiated in a light-dark cycle of time (light condition). The infectious titer of the virus suspension thus placed under each condition was determined 18 days after the start of storage.
The changes were measured by the limiting dilution method on days 39, 83, and 83, respectively, and the changes were shown in the graph (FIG. 3).

As a result, all the samples placed under the light condition (L) and the samples placed at 15 ° C. or more under the dark condition (D) had infectious titers of 18 days after the start of storage. It fell below the detection limit. This result indicates that refrigerated storage in the dark is effective in maintaining the infectivity of the virus for a short period of time, but is not suitable for long-term storage.

Example 7 The following experiment was carried out in order to examine the method of cryopreservation of heterosigma acaciovirus.
SWM3 medium, glycerol or dimethylsulfoxide (DMSO) was added to the HaV suspension whose infectious titer was previously measured by the limiting dilution method so that the volume became 10% by volume, and the solution was dispensed into vials, and then -20 ( Freeze at -80 (deep freezer), -196 ° C (in liquid nitrogen). A sample frozen in this state for 14 days was taken out and thawed in running water until a rice-sized ice block remained. Further, FIG. 4 shows the results of measuring the infectious titer of each of them by the limiting dilution method after sufficiently stirring them. The abbreviations shown on the left of the graph in FIG.
(G) shows the glycerol added group, and (D) shows the DMS
O-added groups are shown. The infectious titer was detected as-
Only the glycerol-added group and the DMSO-added group stored at 196 ° C, the infectious titer of the DMSO-added group was about two orders of magnitude higher than that of the glycerol-added group. In all other experimental plots, the infectious titer was below the detection limit. These results indicate that the addition of 10% DMSO followed by cryopreservation in liquid nitrogen can reduce the loss of HaV infectivity.

Example 8 The concentration of DMSO to minimize the degree of loss of infectivity during the cryopreservation of heterosigma acaciovirus was examined by the following method. DMSO was added to the HaV suspension whose infectious titer was measured in advance by the limiting dilution method so that the volume ratio became 0, 5, 10, 20, and 30% by volume. Frozen. After being stored for 14 days in this state, it was thawed according to the method of Example 8, and the infectious titer of the HaV suspension under each storage condition was measured by the limiting dilution method. FIG. 5 shows the result.
As a result, DMSO was added to the heterosigma acaciovirus suspension as a cryoprotectant to a final concentration of 10-20%, and stored in liquid nitrogen to reduce the infectivity of the heterosigma acaciovirus. It became clear that it can be preserved and retained. FIG. 6 shows the change in the infectious titer when the HaV suspension to which DMSO having a final concentration of 10% was added was stored for a long time. So far, it has been clarified that cryopreservation under these conditions enables cryopreservation while maintaining high infectivity for at least 109 days.
By inoculating HaV regenerated in this manner into a culture of heterosigma acacio artificially grown to a red tide state, it was possible to make the brown turbid culture transparent in about 2 days. (FIG. 7). It should be noted that the sixth embodiment described above.
The virus suspension and the culture solution of heterosigma algae used in 〜8 were prepared according to the methods described in Examples 2 and 1, respectively.

[0043]

Industrial Applicability According to the present invention, there is provided a virus capable of specifically infecting and multiplying algae of the genus Heterosigma. The present invention also provides a method for preserving a virus that can specifically infect and multiply heterosigma algae. Further, according to the present invention, there is provided a method for reproducing the virus stored by the above method. By using the virus of the present invention, red tide can be controlled.

[Brief description of the drawings]

FIG. 1 shows the relationship between the culture time and the growth of heterosigma (heterosigma akashio GS95 and heterosigma akashio UR94).

FIG. 2 is a negative staining image of HaV.

FIG. 3 shows the effect of light and temperature conditions on HaV infectivity.

FIG. 4 shows that after adding various cryoprotectants, -20, -8
The infectivity titer of the HaV suspension cryopreserved at 0, -196 ° C for 14 days is shown.

FIG. 5 shows the infectivity titers of HaV suspensions that were cryopreserved at −196 ° C. for 14 days after adding DMSO at each concentration as a cryoprotectant.

FIG. 6 shows the addition of 10% DMSO as a cryoprotectant,
[Fig. 4] Fig. 4 shows the daily change of the infectious titer of the HaV suspension stored frozen at -196 ° C.

FIG. 7 In the photograph of FIG. 7, the left flask is a heterosigma acacio culture artificially grown to red tide, and the right flask is a dead heterosigma two days after inoculation with HaV. It is a culture of Acacio.

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[Submission date] April 16, 1998

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Claims (13)

[Claims] 1. A virus capable of specifically infecting and multiplying an algae of the genus Heterosigma. 2. The virus according to claim 1, wherein the algae of the genus Heterosigma is Heterosigma akashio. 3. Lack of tail structure and outer membrane structure, particle size 20
3. The virus according to claim 2, wherein the virus is a icosahedron of 2 ± 6 nm and has double-stranded DNA. 4. A liquid sample containing an algae of the genus Heterosigma, which is infected with a virus capable of specifically infecting and amplifying an algae of the genus Heterosigma, is filtered through a filter having a pore size of 0.2 μm. Inoculating and culturing the culture of the heterosigma algae, and cloning the virus by limiting dilution of the culture broth in which the lysates of the heterosigma algae were observed in the culture of the heterosigma algae, A method for isolating a virus capable of specifically infecting and multiplying an alga of the genus Heterosigma. 5. The isolation method according to claim 4, further comprising a step of irradiating the liquid sample with ultraviolet rays. 6. A red tide controlling agent containing, as an active ingredient, a virus capable of specifically infecting and multiplying heterosigma algae. 7. A method for controlling red tide, which comprises spraying a virus capable of specifically infecting and growing on algae of the genus Heterosigma in red tide waters. 8. A method for preserving a virus capable of specifically infecting and multiplying an algae of the genus Heterosigma, wherein the liquid containing the virus is frozen in the presence of dimethyl sulfoxide. . 9. A liquid containing a virus capable of specifically infecting and multiplying Heterosigma algae is frozen in liquid nitrogen in the presence of dimethyl sulfoxide at a concentration of 10 to 20% by volume. The described method. 10. A virus suspension capable of specifically infecting and multiplying Heterosigma algae is prepared and inoculated into a fresh culture solution of Heterosigma algae. 9. The method according to claim 8, comprising a step of mixing the culture supernatant with a dimethylsulfoxide-containing medium and freezing the mixture in liquid nitrogen. 11. A fresh culture solution of an algae of the genus Heterosigma is inoculated with a virus suspension in an amount of 1/100 to 1/50% by volume thereof. ~ 40% by volume
The method according to claim 9 or 10, comprising a step of mixing equal amounts of a dimethyl sulfoxide-containing SWM3 medium at a concentration of 1% and freezing the mixture in liquid nitrogen at -196 ° C. 12. A method for regenerating a virus which is capable of specifically infecting and multiplying an algae of the genus Heterosigma, which is preserved by the method of claim 8, wherein the frozen solution containing the virus is thawed, The above method, wherein a fresh culture solution is inoculated. 13. The method according to claim 12, wherein a fresh culture solution of the algae of the genus Heterosigma is inoculated with a thawed virus-containing solution in an amount of 1% by volume or less.