JP4668900B2 - A method for screening a sample having an antiviral activity against a virus infecting an organism having an acquired immune mechanism using an individual organism having only the innate immunity mechanism or cultured cells thereof, and the antiviral activity using only the innate immunity mechanism Method of evaluating using living organism individual or cultured cells thereof - Google Patents

Description

  The present invention relates to a method for screening a sample having antiviral activity against a virus infecting an organism having an acquired immune mechanism using an individual organism having only the innate immune mechanism or a cultured cell thereof, and an organism having the acquired immune mechanism. The present invention relates to a method for evaluating an antiviral activity against a virus that infects a living organism using only an innate immune mechanism or cultured cells thereof.

  The present inventor has already reported that an antibacterial activity evaluation system for bacteria infecting organisms having acquired immune mechanisms such as humans has been developed using organisms having only innate immune mechanisms such as silkworm larvae. . Specifically, we report that silkworm larvae are infected and killed by pathogenic bacteria against humans such as Staphylococcus aureus, and that antibiotics that are effective in human clinical practice also have therapeutic effects in silkworm larvae. Have proposed the usefulness of silkworm larvae as an evaluation system for therapeutic effects of antibiotics (Non-patent Document 1, Patent Document 1). In addition, antibiotics that are effective for human infections have shown therapeutic effects even in silkworm larvae (Non-Patent Document 2).

  The important point revealed by the above research is that the presence or absence of antibacterial in vitro bacterial growth inhibitory effect differs from the therapeutic effect at the individual level of silkworm larvae. When the former is indicated by MIC (minimum growth inhibitory concentration) and the latter is indicated by ED50 (therapeutically effective dose), the ratio of both (ED50 / MIC) is below a certain value without exception for antibacterial drugs effective in human clinical practice. It was. On the other hand, it is widely known that most of the synthetic compounds obtained by random screening do not show a therapeutic effect even if the antibacterial effect in the test tube is clear. These results indicate that not only the antibacterial activity in vitro but also the pharmacokinetics of the drug in silkworm larvae affects the therapeutic effect. The result that the ED50 values of antibiotics effective in human clinical practice are the same between silkworm larvae and mammals (Non-patent Document 2) is common between silkworm larvae and mammals in terms of drug pharmacokinetics. Suggests that there is a face.

  It has been considered that the therapeutic effect of antiviral drugs using invertebrates cannot be evaluated. One reason for this is that viruses that infect humans have host specificity and are not infectious to invertebrates.

WO2001 / 086287 Kaito et al. Silkworm larvae as an animal model of bacterial infection pathogenic to humans Microbial Pathogenesis 32, 183-190 (2002) Hamamoto et al. Quantitative evaluation of the therapeutic effects of antibiotic by using silkworms infected with human pathogenic microorganisms. Antimicirob. Agents Chemother. 48, 774-779 (2004)

  The present invention relates to a method for screening a sample having antiviral activity against a virus that infects an organism having an acquired immune mechanism, using the individual organism having only the innate immune mechanism or its cultured cells, and evaluation of the antiviral activity. It aims to provide a method.

  The present inventor has obtained the result that the death of the silkworm larvae caused by the nuclear polyhedrosis virus is treated with ganciclovir, foscarnet, vidarabine and ribavirin, which are used as therapeutic agents for cytomegalovirus infection in human clinical practice. This is the first time that antiviral drugs used in human clinical practice have been shown to suppress infection death in invertebrate individuals.

  The inventor has found that by using an individual organism having only an innate immunity mechanism such as a silkworm larva or a cultured cell thereof, an antiviral activity against a virus that infects an organism having an acquired immunity mechanism such as a human is obtained. It is shown that screening of samples having the same and evaluation of the antiviral activity can be performed.

That is, the present invention relates to screening of a sample having an antiviral activity against a virus that infects an organism having an acquired immune mechanism and evaluation of the antiviral activity using an individual organism having only the innate immunity mechanism or cultured cells thereof. More specifically, the following [1] to [12] are provided.
[1] A method for evaluating the presence or absence of antiviral activity of a test sample against a virus that infects an organism having an acquired immune mechanism,
(A) contacting a virus that infects an organism having only the innate immunity mechanism and a test sample with cultured cells of the organism having only the innate immunity mechanism;
(B) a step of detecting an infectious symptom or survival degree of a cultured cell of an organism having only the innate immune mechanism, and (c) when the test sample is not administered (control) And determining whether or not to improve the infectious symptoms of cultured cells of an organism having only the innate immune mechanism or to improve the degree of survival.
[2] A method for evaluating the presence or absence of antiviral activity of a test sample against a virus that infects an organism having an acquired immune mechanism,
(A) a step of administering a virus that infects an organism having only an innate immune mechanism and a test sample to an individual organism having only the innate immune mechanism;
(B) detecting an infectious symptom or the degree of survival of an individual having only the innate immune mechanism, and (c) compared to a case where the test sample is not administered (control), Determining whether or not to improve the infectious symptoms of an individual having only an innate immune mechanism or to improve the degree of survival.
[3] A method for evaluating the presence or absence of antiviral activity of a test sample against a virus that infects an organism having an acquired immune mechanism, comprising the following steps.
(A) Step (b) for evaluating the presence or absence of antiviral activity of a test sample by the method described in [1]. (2) About the sample evaluated as having antiviral activity in step (a) [4] A method for evaluating the presence or absence of antiviral activity of a test sample against a virus that infects an organism having an acquired immune mechanism, comprising the following steps.
(A) contacting a virus that infects an organism having an acquired immune mechanism and a test sample with a cultured cell of the organism having the acquired immune mechanism (b) an infectious symptom of the cultured cell of the organism having the acquired immune mechanism or Step of detecting the degree of survival (c) Whether or not the test sample improves the infectious symptoms of cultured cells of the organism having the acquired immune mechanism as compared with the case where the test sample is not administered (control) Or (d) determining whether or not to improve the degree of survival, it is determined in step (c) that the infectious symptoms of cultured cells of the organism having the acquired immune mechanism are improved or the degree of survival is improved. A step of evaluating the presence or absence of antiviral activity by the method according to [1],
(E) A step of evaluating the presence or absence of antiviral activity by the method according to [2] for the sample evaluated to have antiviral activity in step (d) [5] An acquired immune mechanism comprising the following steps: A method for screening a sample having antiviral activity against a virus that infects a living organism.
(A) A step of evaluating the presence or absence of antiviral activity against a virus infecting an organism having an acquired immune mechanism for a plurality of test samples by the evaluation method according to any one of [1] to [4] (b) A step of selecting, from a plurality of test samples, a sample evaluated to have antiviral activity against a virus that infects an organism having an acquired immune mechanism. [6] The organism having an acquired immune mechanism is a mammal. The method according to any one of [1] to [5].
[7] The method according to [6], wherein the mammal is a human.
[8] The method according to any one of [1] to [7], wherein the organism having only the innate immune mechanism is an invertebrate larva.
[9] The method according to [8], wherein the larva is large.
[10] The method according to [8] or [9], wherein the invertebrate is an insect.
[11] The method according to [10], wherein the insect is a silkworm.
[12] The organism having only the innate immune mechanism is a silkworm larva, and the virus is a nuclear polyhedrosis virus, a cytoplasmic polyhedrosis virus, a softening disease virus, or a dense nucleus disease virus, [1] to [7] The method according to any one.

It is a graph which shows the infection death amount of the silkworm larva by a nuclear polyhedrosis virus. Nuclear polyhedrosis virus load was quantified by plaque formation in BmN cells. Each amount of virus was injected into the blood of 5th instar silkworm larvae (10 per group) and the decrease in survival rate was recorded. It is a photograph which shows the therapeutic effect with respect to the infection death of the silkworm larva by the nuclear polyhedrosis virus of a ganciclovir and a foscarnet. Injection of 4 x 10 ⁴ pfu nuclear polyhedrosis virus into the blood of silkworm fifth instar larvae, physiological saline (second from the left) ganciclovir (0.5 mg, second from the right) or foscarnet (2.5 mg, right end) was injected, and 120 hours later was photographed. A control (left end) was injected with a culture solution and physiological saline instead of the virus solution. It is a graph which shows the time course of the survival rate fall of a silkworm larva. Infect silkworm 5th instar larvae with 4 x 10 ⁴ pfu of nuclear polyhedrosis virus, and then inject physiological saline, ganciclovir (0.5mg) or foscarnet (2.5mg) to reduce the survival rate of silkworm larvae. Recorded. It is a graph which shows suppression of the increase in the virus particle in the silkworm larva body by an antiviral agent. After injecting 5x10 ⁴ pfu nuclear polyhedrosis virus into 5th instar larvae, and then injecting physiological saline, ganciclovir (1 mg), or holcarnet (2.5 mg), vidarabine (2 mg), ribavirin (1 mg) On the day, blood was collected from the larvae, plaque assay was performed, and the number of viruses per mL of blood was determined. The experiment was performed independently for each drug. The white bar is the control group injected with physiological saline, and the black bar is the group injected with drug. It is a graph which shows suppression of the nuclear polyhedrosis virus growth of an antiviral agent in the silkworm larva origin cultured cell. 1 × 10 ⁵ BmN4 cells and 1 × 10 ⁵ viruses were added to a 24-well culture dish, and antiviral drugs were added at various concentrations, followed by culturing for 5 days. Cell culture medium was collected, plaque assay was performed, and the number of viruses per mL was counted. It is the figure which showed ED50 value with respect to the nuclear polyhedrosis virus infection silkworm larva of an antiviral agent, and IC50 value in a cultured cell system. IC50 indicates the concentration of the antiviral agent in the culture solution necessary for the virus growth to be suppressed to 50% as compared with the case where the antiviral agent is not added. It is the graph which showed the therapeutic effect with respect to the nuclear polyhedrosis virus infection silkworm larva of a Chinese medicine. 1.6 x 10 larvae injected with ⁴ nuclear polyhedrosis viruses were further administered with 100 mg Mao-to, Sho-saiko-to 200 mg, and Kakkon-to 200 mg in the intestine, and the survival rate was measured over time.

  The present invention relates to a method for screening a sample (candidate compound) having antiviral activity against a virus infecting an organism having an acquired immune mechanism, using an individual organism having only innate immunity or cultured cells thereof, and the antivirus. Provided is a method for evaluating an activity using an organism having only innate immunity or a cultured cell thereof.

Examples of a method for evaluating the presence or absence of antiviral activity of a test sample against a virus that infects an organism having an acquired immune mechanism include a method including the following steps.
(A) A step of administering a virus that infects an organism having only the innate immunity mechanism and a test sample to the individual organism having only the innate immunity mechanism (b) Infectious symptoms or survival of the individual organism having only the innate immunity mechanism (C) whether or not the test sample improves the infectious symptoms of an individual having only the innate immune mechanism, as compared with the case where the test sample is not administered (control), or Step of determining whether or not to improve the degree of survival

  In the above method, when the test sample improves the infectious symptom of an organism individual having only the innate immune mechanism or improves the degree of survival compared to the control, the test sample exhibits the acquired immune mechanism. It is evaluated to have antiviral activity against viruses that infect living organisms. A sample that is evaluated to have antiviral activity by an evaluation method that uses an individual organism having only an innate immune mechanism is highly likely to exert a therapeutic effect in clinical practice, and is a promising candidate for an antiviral drug.

  An evaluation method using an individual organism having only an innate immune mechanism includes (1) search for an antiviral drug using as an index the therapeutic effect on the virus infection death of an organism having only the innate immune mechanism, and (2) as described later. The present invention can be used for a therapeutic effect test on a sample that has been shown to have an inhibitory effect on virus growth in a cell culture system.

  Targeting mechanisms essential to the survival of viruses common to viruses that infect organisms that have acquired immune mechanisms and viruses that infect only organisms that have only innate immunity mechanisms by an evaluation method that uses individuals with only innate immune mechanisms Search for antiviral drugs.

  In the present invention, the “innate immunity mechanism” means an immune defense mechanism (innate immunity mechanism) that does not depend on an acquired immunity (acquired immunity) mechanism. Vertebrates have an acquired immune mechanism that protects the living body using molecules that specifically recognize invaders such as antibodies against the invasion of pathogens.Invertebrates and plants have such an acquired immune mechanism. do not do. In other words, the “organism having only the innate immune mechanism” in the present invention is an invertebrate and a plant having no acquired immune mechanism.

  In the present invention, the organism to which the virus is administered is not particularly limited as long as it has only the innate immune mechanism, but an organism belonging to insects is a preferred example. In the present invention, the term “insects” refers to a class of arthropods from the greater submaxillary phylum, consisting of the four subclasses of caterpillars, flying beetles, wormless insects, and rodents. . There is no restriction | limiting in particular as an organism which belongs to the insects used for this invention. From the convenience of handling, larvae are preferable.

  From the viewpoint of ease of administration of virus or test sample, the larvae are preferably large. In the present invention, the term “large larva” refers to a larva having a body length of 1 cm or more. Examples of organisms other than insects include, for example, arthropods other than insects such as spiders and scorpions, molluscs such as slugs and snails, annelids such as earthworms, cynomolgus animals such as starfish and sea urchins, worms, roundworms, etc. And other plants such as linear animals such as hydra, sea anemone, jellyfish, etc., rice, radish, etc., and these organisms are also contemplated for use in the present invention.

  Examples of the larvae in the present invention include, but are not limited to, Lepidoptera (including moths and butterflies) and Coleoptera (including beetles). The larvae are preferably silkworms (which belong to the order of arthropoda, submaxillary, arachnids, butterflies) that have a fast generation change, can be reared easily in the laboratory, and have been genetically analyzed. . Silkworms have large larvae, making it easier to inject pathogens and drugs compared to small organisms such as C. elegans (belonging to the order of C. elegans, Nematoda, Nematoda subfamily, Coleoptera) (Okada, E. et al. 1997. J. Seric. Sci. Jpn. 66: 116-122.). For this reason, it is thought that it is very suitable for the evaluation of the antibacterial agent with respect to a pathogen. Moreover, the use of silkworm larvae has no ethical problem and is highly useful compared to mammals.

  It has been found that the pharmacokinetics of compounds greatly influences the therapeutic effect of pharmaceuticals. However, in general, investigating whether there is a therapeutic effect based on pharmacokinetics at the initial stage of the search has a problem in terms of cost. Silkworm larvae are much cheaper to handle than mammals such as monkeys. Therefore, by using silkworm larvae, it is expected that it is possible to determine the presence or absence of a therapeutic effect of a test sample against virus infection death at an early stage of searching for an antiviral drug.

  In the development of antiviral drugs so far, candidate compounds are selected based on the inhibition of virus growth in human cell culture systems, and compounds with low concentrations (IC50) necessary for virus growth inhibition are used in infection models such as monkeys. The therapeutic effect has been evaluated. Therefore, there has been a problem that the cost required for animal experiments becomes enormous. If silkworm larvae are used, it is considered that a substance having good dynamics in the individual of the candidate compound can be selected.

  According to the inventor's research on the search for antibacterial drugs using silkworm larvae, most of the compounds showing antibacterial activity in vitro do not show a therapeutic effect. These compounds are considered to have no therapeutic effect due to poor pharmacokinetics. On the other hand, none of the antibacterial drugs that are effective in treating infections in clinical humans have an ED50 / MIC ratio of 10 or more, and all of them showed high therapeutic effects in silkworm larvae. This result shows that in the system using silkworm larvae, compounds effective in human clinical practice are not dropped in the search process. In the search for antiviral drugs, it is necessary to select a compound having good pharmacokinetics and showing a therapeutic effect. Therefore, the silkworm larvae system is considered to be effective.

  Silkworm larvae are of a size that is convenient to handle by hand, and because they do not move much, injection of viruses and drugs is very easy. Skilled researchers can process more than 100 silkworm larvae per hour. The inventor's experience is simpler than examining the antiviral effects of candidate compounds in cultured cell lines in vitro. In addition, the toxicity of the candidate compound to an individual can be easily evaluated.

  In addition, when silkworm larvae are used, screening costs for antiviral drugs and evaluation of antiviral activity are greatly reduced compared to the case of using conventional mammals. It becomes possible.

  In the present invention, “a virus that infects an organism having only an innate immunity mechanism” means a virus that infects an organism having at least an innate immunity mechanism. Therefore, the “virus that infects an organism having only an innate immune mechanism” includes a virus that infects both an organism having only an innate immune mechanism and an organism having an acquired immune mechanism. Further, “a virus that infects an organism having only the innate immunity mechanism” is a virus that infects at least one organism having only the innate immunity mechanism. The type of “virus that infects an organism having only an innate immune mechanism” is not particularly limited, and may be a DNA virus or an RNA virus. When a silkworm larva is used as an organism having only an innate immune mechanism, a silkworm nuclear polyhedrosis virus, a cytoplasmic polyhedrosis virus, a softening disease virus, and a dense nucleus disease virus can be exemplified, but not limited thereto.

  In an evaluation method using an individual organism having only an innate immune mechanism, if a DNA virus is used as a virus that infects an organism having only the innate immune mechanism, an antiviral agent against the DNA virus infecting an organism having an acquired immune mechanism, or If an RNA virus is used as a virus that infects an organism having only the innate immune mechanism, it is possible to at least search for an antiviral drug against the RNA virus that infects an organism having an acquired immune mechanism.

  In the present invention, the “organism having an acquired immune mechanism” is preferably a fish such as a carp, a bird such as a chicken, a mammal such as a mouse, rat, rabbit, dog, cat, monkey or the like, more preferably a human. is there.

  In the present invention, a preferred embodiment of “a virus that infects an organism having an acquired immune mechanism” is a virus having the ability to infect a host and cause a disease. For example, DNA viruses such as cytomegalovirus and herpes virus, and AIDS RNA viruses such as, but not limited to, retroviruses such as viruses, orthomyxoviruses such as influenza viruses, coronaviruses such as SARS, picornaviruses such as poliovirus, and arenaviruses including Lassa virus is not.

  In the present invention, the test sample is not particularly limited, and a desired sample to be evaluated for antiviral activity is used. Examples of test samples include cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, purified or crude proteins, peptides, non-peptidic compounds, synthetic low molecular compounds, natural Examples include, but are not limited to, compounds and compound libraries.

  For example, intraperitoneal administration, injection into blood, addition to feed (food), intestine, virus infecting organisms having only the innate immunity mechanism and administration of test samples to individual organisms having only the innate immunity mechanism It can be performed by a method such as injection into the inside.

  The dose of a virus that infects an organism having only the innate immunity mechanism or the individual organism having only the innate immunity mechanism of the test sample varies depending on the virus, the host, the type of the test sample, and the like. In general, the virus is administered in an amount of about 100 to 10,000 p.f.u. (plaque forming unit). When larvae of organisms belonging to insects are used as a host, for example, an amount of about 100 to 10,000 p.f.u. may be injected into the blood from the back.

  For the test sample, the minimum amount for killing an individual organism having only the innate immune mechanism to be used or its cultured cells is determined, and an amount less than that is administered. A person skilled in the art will be able to select an appropriate dose according to the virus that infects an organism having only the innate immunity mechanism, the organism having only the innate immunity mechanism, the type of test sample, and the like.

  Infectious symptoms to be detected include, for example, (i) an increase in the number of viruses in an individual having only an innate immune mechanism (hereinafter referred to as a host), (ii) a decrease in host weight or an increase in host weight. Inhibition, (iii) reduction in the amount of antiviral substances in the blood of the host, (iv) failure of the host's immune function, (v) reduction of various enzyme activities in the body fluids and organs of the host, and the like. If the host is an insect larva, for example, it may be detected that it does not molt into an old larva or become a pupa or adult. In the present invention, the degree of survival of the host may be detected in addition to the above infection symptoms. Examples of the degree of survival include survival rate and survival period.

Moreover, as an evaluation method of this invention, the method including the following processes is mentioned.
(A) a step of contacting a virus that infects an organism having only the innate immunity mechanism and a test sample with a cultured cell of the organism having only the innate immunity mechanism; and (b) a culture cell of the organism having only the innate immunity mechanism. Step (c) of detecting infectious symptoms or degree of survival (c) Compared with the case where no test sample is administered (control), the test sample improves the infectious symptoms of cultured cells of an organism having only the innate immune mechanism Determining whether or not to improve the degree of survival

  In the above method, when the test sample improves the infectious symptoms of the cultured cells of the organism having only the innate immune mechanism or improves the degree of survival compared to the control, the test sample acquires the acquired immunity. It is evaluated to have antiviral activity against viruses that infect organisms with mechanisms.

  In the present invention, as a method of contacting a virus that infects an organism having only the innate immunity mechanism and a test sample with a cultured cell of the organism having only the innate immunity mechanism, for example, the virus and the test sample are added to the culture solution of the cultured cell. This can be done by adding a test sample. When the test sample is a protein, for example, a vector containing a DNA encoding the protein can be introduced into cultured cells.

  Culture cells well known to those skilled in the art can be used as cultured cells of organisms having only the innate immune mechanism. For example, BmN cells can be used as long as they are cultured cells derived from silkworm larvae.

  Examples of infectious symptoms to be detected include an increase in the number of viruses in cultured cells of an organism having only an innate immune mechanism. In the present invention, the degree of survival of the cultured cells may be detected by using a decrease in mitochondrial metabolic activity of the cultured cells by MTT assay or the like as an index. Examples of the degree of survival include survival rate and survival period.

  In the present invention, the presence or absence of antiviral activity of the test sample is first evaluated using cultured cells of the organism having only the innate immunity mechanism, and then an individual organism having only the innate immunity mechanism is selected. It is also possible to evaluate the presence or absence of antiviral activity of a sample evaluated as having antiviral activity. By such a method, it can be evaluated more accurately whether a test sample is a candidate of an antiviral drug applicable clinically. When the purpose is to search for an antiviral drug, either one of a step of using a cultured cell of an organism having only an innate immune mechanism and a step of using an individual of an organism having only an innate immune mechanism. It may be omitted.

Furthermore, in this invention, it is also possible to evaluate the presence or absence of antiviral activity of a test sample by a method including the following steps.
(A) contacting a virus that infects an organism having an acquired immune mechanism and a test sample with a cultured cell of the organism having the acquired immune mechanism (b) an infectious symptom of the cultured cell of the organism having the acquired immune mechanism or Step of detecting the degree of survival (c) Whether or not the test sample improves the infectious symptoms of cultured cells of the organism having the acquired immune mechanism as compared with the case where the test sample is not administered (control) Or (d) determining whether or not the degree of survival is to be improved In step (c), it is determined that the infectious symptoms of cultured cells of the organism having the acquired immune mechanism are improved or the degree of survival is improved. The sample is evaluated to have antiviral activity in step (e) and step (d) in which the presence or absence of antiviral activity of the test sample is evaluated using cultured cells of organisms having only the innate immune mechanism. The sample The step of evaluating the presence or absence of antiviral activity using an individual organism having only an innate immune mechanism is highly likely that a sample evaluated as having antiviral activity will exert a therapeutic effect in clinical practice. A potential candidate for antiviral drugs.

  In the steps (a) to (c), the organism having the acquired immune mechanism and the type of virus that infects the organism are not particularly limited. In the present invention, for example, a test sample is brought into contact with a human cultured cell with a desired virus that infects humans, and the test sample improves the infection symptoms of the human cultured cell by a method well known to those skilled in the art. Whether or not to improve the degree of survival can be determined.

  Although the specific example of the said method is shown below, it is not limited to this method. Examples of the method include (1) a step of obtaining the antiviral action of a test sample against human virus as IC50a in a virus propagation system using cultured human cells, and (2) silkworm nuclear polyhedrosis in the sample. Antiviral activity against viruses is determined as IC50b in a nuclear polyhedrosis virus propagation system using silkworm larvae cultured cells (for example, BmN cells). (3) Infection of nuclear polyhedrosis virus with silkworm larvae in the sample And a method including a step of obtaining a death inhibitory effect as ED50. A low IC50a value is important as a criterion for determining the action of the compound on the target virus. Furthermore, the value of ED50 / IC50b is a standard for judging the therapeutic effect of the compound in the silkworm larva system. The smaller this value, the better the pharmacokinetics of the silkworm larvae of the test sample and the higher the therapeutic effect.

In the present invention, by utilizing the above-described various evaluation methods, the presence or absence of antiviral activity against a virus that infects an organism having an acquired immune mechanism is evaluated for a plurality of test samples. It is also possible to select samples that have been evaluated as having antiviral activity against the infecting virus. The present invention also provides a method for screening a sample having antiviral activity against a virus that infects an organism having such an acquired immune mechanism.
In addition, all prior art documents cited in the present specification are incorporated herein by reference.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to a following example.
The titer of nuclear polyhedrosis virus (mutant that mutated the nuclear polyhedrogenesis gene and became unable to orally infect) was measured by plaque-forming ability on soft agar using silkworm larvae-derived cultured cells BmN .

  Silkworm larvae (Hu ・ Yo x Tukuba ・ Ne) purchase eggs from Ehime species, give silk mate 2S (Japanese agricultural products), grow to 4 years old sleep state, leave for 1-3 days, then feed again One day later, 0.05 ml of the nuclear polyhedrosis virus solution was injected into the blood with a Terumo syringe (1 mL). Further, 0.05 ml of an antiviral drug dissolved in 0.6% NaCl was subsequently injected into the blood. The survival rate of silkworm larvae was observed continuously for at least 5 days.

1. Infection and death of silkworm larvae caused by nuclear polyhedrosis virus Figure 1 shows changes in the survival rate of silkworm larvae when the nuclear polyhedrosis virus is injected into the blood of silkworm larvae. Infection death of silkworm larvae depending on the viral load was observed. In addition, since 40p.fu virus also causes infection death, it is thought that in this system, infection death is caused by a small number of virus particles.

2. Therapeutic effects of ganciclovir and foscarnet on the death of silkworm larvae caused by nuclear polyhedrosis virus Ganciclovir and foscarnet are nucleic acid derivatives and phosphate analogs, respectively. The former is phosphorylated by thymidine kinase encoded by a viral gene to become a substrate for DNA synthesis, and as a result, inhibits DNA synthesis. The latter is also an inhibitor of viral DNA polymerase. These are used clinically as therapeutic agents for infections caused by human cytomegalovirus and herpes virus. As shown in Figures 2 and 3, deaths caused by intra-blood injection of 4 x 10 ⁴ pfu nuclear polyhedrosis virus are injected with 0.5 mg ganciclovir or 2.5 mg foscarnet per silkworm larvae. This was clearly suppressed. 120 hours after virus injection, the total number of silkworm larvae died, but the antiviral dose (ED50) at which 50% survived was 0.05 mg / g for ganciclovir and 0.6 mg / focalnet for foscarnet. g. Larva. Vidarabine was 0.145 mg / g · larvae, and ribavirin was 0.01 mg / g · larvae. These values are 4-10 times the human therapeutic dose.

3. Inhibition of virus growth in silkworm larvae by antiviral drugs As shown in FIG. 4, the growth of viruses in silkworm larvae is suppressed by administration of antiviral drugs. Therefore, suppression of infection death in silkworm larvae caused by the nuclear polyhedrosis virus, an antiviral drug, is thought to be due to suppression of virus growth in the body.

4). Antiviral drug growth suppression in silkworm larvae-derived cultured cells As shown in FIG. 5, the growth of viruses infected with cultured cells is suppressed depending on the concentration of the antiviral drug. In this system, the concentration at which inhibition of virus growth was suppressed to 50% compared to the case without antiviral agent was taken as IC50, and the ED50 / IC50 value was calculated to be 3.0 or less as shown in FIG. This value is a reference value in evaluating the therapeutic effect of a novel antiviral drug.

5. Therapeutic effect of Kampo medicines on silkworm larvae infected with nuclear polyhedrosis virus Anti-viral effects on herbal larvae infected with nuclear polyhedrosis virus infection It was investigated. As shown in FIG. 7, it was found that among the three Chinese herbal medicines examined, Mao-to, Sho-saiko-to, and Kakkon-to, Mao-to has a life-prolonging effect. Furthermore, of the herbal medicines that make up Mao-to, Keihi was found to have a life-prolonging effect on the infection of silkworm larvae caused by the nuclear polyhedrosis virus. These results indicate that unknown antiviral drugs can be discovered using the present invention.

  It has been considered impossible to evaluate the therapeutic effect of antiviral drugs using organisms having only the innate immune mechanism such as invertebrates. The present inventor, based on such common technical knowledge, uses a living individual having only an innate immune mechanism or a cultured cell thereof, a sample having antiviral activity against a virus that infects an organism having an acquired immune mechanism. It has been found for the first time in the world that screening and evaluation of the antiviral activity can be performed.

  According to the present invention, a virus that does not infect an organism having an acquired immune mechanism, but does not infect an organism having an acquired immune mechanism, even when an infectious virus is used for an organism having only an innate immune mechanism. Antiviral drugs targeting a mechanism common to viruses that infect organisms having only an immune mechanism can be searched. Furthermore, according to the present invention, it is possible to evaluate the therapeutic effect of a test sample on virus infection death at the individual level.

  Among the methods of the present invention, by using a method utilizing an individual organism having only an innate immune mechanism, it is possible to more effectively and effectively apply an anti-antibody that can be clinically applied as compared with a method utilizing a conventional human cultured cell system. Viral drug candidate compounds can be isolated and identified. In addition, a living individual having only an innate immune mechanism or a cultured cell thereof used in the method of the present invention is lower in cost and easier to handle than an infection model such as a monkey used in a conventional method. . That is, according to the present invention, it is possible to provide a low-cost and simple screening method and evaluation method for candidate compounds of antiviral drugs.

Claims (11)

A method for evaluating the presence or absence of antiviral activity of a test sample against a virus that infects an organism having an acquired immune mechanism,
(A) administering a virus infecting silkworm larvae and a test sample to the silkworm larvae,
(B) detecting an infectious symptom or the degree of survival of the silkworm larva individual,
(C) Compared with the case where the test sample is not administered (control), it is determined whether or not the test sample improves the infectious symptoms of the silkworm larva individual or increases the degree of survival. The process of
(D) The step of improving the infectious symptom of the silkworm larva individual in step (c) or bringing the test sample to improve the degree of survival into contact with a cultured cell of an organism having the acquired immune mechanism,
(E) a step of detecting an infectious symptom or degree of survival of cultured cells of an organism having the acquired immune mechanism, and (f) when the test sample is not administered (control), Determining whether to improve the infectious symptoms of cultured cells of the organism having the acquired immune mechanism or to improve the degree of survival;
Including methods. A method for evaluating the presence or absence of antiviral activity of a test sample against a virus that infects an organism having an acquired immune mechanism, comprising the following steps.
(A) contacting a virus that infects an organism having an acquired immune mechanism and a test sample with a cultured cell of the organism having the acquired immune mechanism (b) an infectious symptom of the cultured cell of the organism having the acquired immune mechanism or Step of detecting the degree of survival (c) Whether or not the test sample improves the infectious symptoms of cultured cells of the organism having the acquired immune mechanism as compared with the case where the test sample is not administered (control) Or (d) determining whether or not the degree of survival is to be improved In step (c), it is determined that the infectious symptoms of cultured cells of the organism having the acquired immune mechanism are improved or the degree of survival is improved. (E) detecting the infection symptoms or the degree of survival of the silkworm larvae, and (f) not administering the test sample (control), The test sample is Determining whether to improve the degree of infection whether ameliorate the symptoms, or survival of the silkworm larvae individual A method for screening a sample having antiviral activity against a virus that infects an organism having an acquired immune mechanism, comprising the following steps.
(A) a step of evaluating the presence or absence of antiviral activity against a virus that infects an organism having an acquired immune mechanism for a plurality of test samples by the evaluation method according to claim 1 or claim 2; A step of selecting a sample evaluated as having antiviral activity against a virus that infects an organism having an acquired immune mechanism from a test sample   The method according to any one of claims 1 to 3, wherein the organism having the acquired immune mechanism is a mammal.   The method of claim 4, wherein the mammal is a human. Virus is a nuclear polyhedrosis virus, cytoplasmic polyhedrosis virus, a softening disease virus or Kokakubyo virus A method according to any of claims 1 to 5. A method for screening whether a candidate antiviral drugs test sample exhibits a therapeutic effect against Shi clinical in viruses that infect humans,
(A) administering a virus infecting silkworm larvae and a test sample to the silkworm larvae,
(B) detecting an infectious symptom or the degree of survival of the silkworm larva individual,
(C) Compared with the case where the test sample is not administered (control), it is determined whether or not the test sample improves the infectious symptoms of the silkworm larva individual or increases the degree of survival. And (d) selecting a test substance that improves an infectious symptom of the silkworm larva individual or increases the degree of survival.
Including methods. A method for screening whether a candidate antiviral drugs test sample exhibits a therapeutic effect against Shi clinical in viruses that infect humans,
(A) a step of administering a virus that infects silkworm larvae and a test sample to the silkworm larva individual, and contacting the silkworm larvae with cultured cells;
(B) detecting an infectious symptom or the degree of survival of the silkworm larva individual and the cultured cells of the silkworm larva,
(C) Compared with the case where the test sample is not administered (control), whether or not the test sample improves the infection symptoms of the silkworm larvae individual and the cultured cells of the silkworm larvae, or the degree of survival. A step of determining whether or not to improve, and (d) ED50 value [μg / (girth of silkworm larvae)] of a test sample against a silkworm larva infected with a virus, and propagation of a virus infected with a cultured cell of the silkworm larva Is a ratio with the concentration IC50 [μg / mL] of the test sample that is suppressed to 50% compared to the case where the test sample is not contacted,
ED50 value [μg / (Silk larva g)] / IC50 [μg / mL]
Selecting a test sample whose value is below a certain value,
The method of claim 7 comprising:   The method according to claim 8, wherein the virus infected with the silkworm larvae and the virus infected with the cultured cells of the silkworm larvae are nuclear polyhedrosis viruses.   The method according to claim 8 or 9, wherein the cultured cells of the silkworm larva are BmN cells.   11. The method according to claim 8, wherein it is determined whether or not the value of the “ED50 value [μg / (Silkworm Larvae g)] / IC50 [μg / mL]” is 3 or less. Method.