KR100682069B1 - Method for screening antiviral agent - Google Patents

Abstract

The present invention relates to a method of measuring the activity of a virus and detecting antiviral agents using sulfurhodamine.

Viral activity, virus assay, antiviral agents

Description

Antiviral agent detection method {Method for screening antiviral agent}             

Figure 1 shows Vero cells infected with porcine epidemic diarrhea at various active concentrations and the remaining cells stained with SRB.

Figure 2 shows Vero cells infected with various active concentrations of swine pandemic diarrhea and examined the morphology of the cells under a microscope.

Figure 3 is a comparison of the percentage of absorbance of virus-infected wells with percentages of the virus-infected cells infected with Vero cells with various active concentrations of porcine epidemic diarrhea and stained with SRB. It is.

Figure 4 is a comparison of the percentage of absorbance of virus-infected wells with percentages of the infection of MDCK cells with various concentrations of human influenza type A virus and stained with SRB, followed by staining with an average value of 100% of cells not infected with virus. It is.

FIG. 5 is a comparison of the absorbance of virus-infected wells with percentages, after MDCK cells were infected with human influenza B virus at various concentrations and stained with SRB, and then the average absorbance of cells not infected with virus was 100%. It is.

Figure 6 shows the percentage of absorbance of virus-infected wells infected with MA104 cells with various concentrations of porcine rotavirus and bovine rotavirus and stained with SRB, and then the average absorbance of cells not infected with virus was 100%. It is a comparative calculation.

FIG. 7 shows Vero cells infected with porcine epidemic diarrhea virus, followed by treatment with ribavirin, acyclovir, and lamivudine at concentrations of 100, 10, 1, and 0.1 μg / ml, respectively.

8 is infected with Vero cells swine epidemic diarrhea virus, and treated with ribavirin, acyclovir, lamivudine at a concentration of 100, 10, 1, 0.1 ㎍ / ㎖, respectively, to examine the ability to inhibit virus growth.

The present invention relates to a method of measuring the activity of a virus and detecting antiviral agents using sulfurhodamine.

Viruses infect organisms and cause disease. Viral infections of plant and livestock infections not only cause significant economic losses, but also cause cancer, immune and respiratory diseases in humans by secondary hepatitis caused by the ingestion of organisms infected with these viruses, and by direct infection with viruses that host humans. It has been suffering for a long time from various diseases such as diarrhea, mental illness, skin disease and conjunctival diseases. Most of the viral infectious diseases are difficult to diagnose early, and as can be seen in AIDS, cancer, SARS, etc., despite the incurable diseases, anti-viral drugs with clear efficacy have not yet been developed. In addition, most viruses lower the efficacy of the vaccine by varying the characteristics of the antigen.

Antiviral agents manufactured and used to date include, but are not limited to, hepatitis C (Foster Semin Liver Dis. 2004), SARS, Ribavirin (Gunther et al. Antiviral Res. 2004), Herpes, which are used to treat infection with Ebola virus. Acyclovir (Dewhurst Herpes, 2004) for the treatment of herpesviruses, and lamivudine (Lai, 2004, J. Gastroenterol. Hepatol) for the treatment of HBV. However, these drugs are not only effective, but also have high side effects. Therefore, there is a need for the development of new antiviral agents with fewer side effects and excellent efficacy.

However, the discovery of new antiviral agents for the treatment of viruses was not easy due to the difficulty of measuring virus activity.

Viruses are the smallest of the organisms, having a size of 20 to 300 μm and having a double or single strand of DNA or RNA as a genome. The nucleic acid of a virus is surrounded by a protein shell and may contain some enzymes. Because viruses cannot replicate their genomes themselves, they infect other organisms and replicate them in host cells. Virus generated in the host cell is released out of the cell, the cell membrane of the host cell is broken in the process of the generated virus to bind the cell membrane. Viruses released out of cells through the disrupted cell membranes infect neighboring cells and, during replication, are released as they break down and infect nearby cells. Through infection, replication, and cell disruption, viruses gradually break down surrounding host cells. By repeating this process, we can observe the disruption of the host cell with eyes, and the host cell disruption is called plaque. If you look at the place where the plaque has occurred, you will not see any cells in the broken part, and there may be cells that are not broken yet. All virus replications go through this process of infection, replication, and crushing, and the time out of the cell from infection to the host cell varies by type of virus.

Viral infections occur when viral surface proteins specifically bind to cell surface proteins. Therefore, even if the same individual, depending on the organ and tissue cells, the viral infection can vary greatly. Even if a virus is infected with a host cell, the virus does not grow unless the environment in the host cell is suitable for virus propagation. Therefore, in order to detect useful host cells for virus culture or to measure the infectivity and activity of the virus, it is necessary to accurately measure the degree of cell disruption by the virus.

To date, the method of measuring the host cell disruption ability of the virus has been relied on plaque assay. Plaque assay is to culture the host cells of the virus to be measured in each well of the 6-well plate for cell culture, and then the culture supernatant is removed and the virus solution diluted to various concentrations is added to infect the virus for a predetermined time, and the phosphate buffer solution is used. The wells are washed to remove uninfected virus. Then, a culture solution containing 1% agarose was added, the agarose culture solution was solidified at 4 ° C. for 10 minutes, and then cultured in a 37 ° C. incubator for 2 to 7 days. When plaque is formed in the well plate, the cells are stained for 2 hours by adding a culture solution containing 0.4% of neutral red to better observe the plaque, and the wells are washed with phosphate buffer. Viral activity is determined by measuring the number of plaques formed according to the concentration of the virus (Natalie et al. 1976. J. Clin. Microvirol.). At this time, the plaque formed value is expressed as pfu (plaque forming unit). However, the form of plaques formed varies depending on the type of virus that is infected and often shows atypical forms. Therefore, the measurement of plaque number is accompanied by many errors by subjective judgment. In addition, plaque measurement is performed in a 6-well plate, so there is a disadvantage that large-volume analysis is impossible. Thus, methods for measuring viral plaques in 96-well plates have been studied. Staining Cells with Neutral Red (Huffman et al. 1997. Antiviral Chem. Chemother.), And Staining Cells with Crystal Violet (Gabrielsen et al. 1992. J. Natural Prod.) Etc. were studied. Plaques can be observed in the wells of 96 well plates through the above methods, but since the results are visually confirmed, there is a disadvantage in that the reliability of the results is low due to the subjective judgment when forming atypical plaques (Waschke and Staber 1976). J. Hyg.Epidemiol.Microbiol.Imunmun.).

To remedy this problem, a method using MTT has been developed (Nakashima et al. 1989. J. Virol Methods). When MTT staining solution was added to the virus-infected cells and reacted for 2 hours, MTT reacted with the enzyme in the mitochondria was precipitated at the bottom of the well, and 1% SDS solution was added thereto to dissolve the precipitated MTT, followed by absorbance with ELISA reader. Measure It is possible to experiment in 96-well plates by measuring the activity of mitochondria, an intracellular organ, in a 96-well plate.However, if the number of cells in the well is less than 150, it cannot be measured (Haselsberger et al. 1996. Anticancer drugs). It has the disadvantage of being affected by substances with antioxidant capacity (Natarajan et al. 2000. Cancer Detect Prev.). In addition, the MTT method uses SDS, which is a strong solubilizer, and thus, there is a disadvantage in that plaque, which is another indicator of virus activity, cannot be observed.

Conventional virus activity measurements have the disadvantages of being costly, time consuming, cumbersome, incapable of processing in large quantities and inaccurately reliant on the results.

On the other hand, sulforhodamine B (SRB) is a substance that binds to cellular proteins, which is less reactive with anticancer drugs than MTT, and is used for the screening of anticancer drugs because the linear curve according to the concentration of anticancer drugs is closer to the straight line. (Rubinstain et al. 1990 J Natl cancer Inst; Perez et al. 1993. Eur. J. cancer; Banasiak et al. 1999. Radiat. Oncol. Invest,). However, no studies of using SRB to measure virus activity have been reported and have not been used in the search for antiviral agents.

Under this background, the present inventors endeavored to develop a method for measuring viral activity that can detect a large amount in a short time through a highly reliable and simple process. When using sulfuridamine, all of the above points are met to effectively measure viral activity. It was confirmed that the antiviral agent can be effectively detected using the same, and completed the present invention.

One object of the present invention is to provide a method for measuring viral activity by infecting a cell to be infected with a virus with a virus and measuring the amount of sulfodadamine that binds to cellular proteins.

Still another object of the present invention is to provide a method for detecting an antiviral agent by infecting a cell to be infected with the virus with a virus, treating the cell with an antiviral agent, and then measuring the amount of sulfodadamine bound to the cellular protein.

In one embodiment, the present invention provides a method of culturing a cell to be infected with a virus; Infecting the cells with a virus; Immobilizing the cells and staining with sulforhodamine; A method of measuring viral activity, comprising measuring the amount of sulfodadamine that binds a cellular protein and comparing it to a control cell that is not infected with a virus.

In the present invention, "viral activity" refers to an activity in which a virus proliferates through an lytic cycle in an infected cell, and induces destruction of the cell when the virus is proliferated in an infected cell and then discharged. This can be seen by measuring the degree of fracture. According to the present invention, sulphide-damine, which has not been previously used for measuring virus activity, has high validity due to accurate measurement results and high reliability of consistent results even in repeated measurements. In vitro cell-based viral activity assays are provided.

Sulfurhodamine is a dye that can bind to basic amino acids of cellular proteins. Taking note of the fact that quantitative measurement of cells is possible by measuring the degree of color development by the binding of sulforhodamine and cellular proteins, quantitatively measuring the degree of cell destruction caused by virus infection compared to the control group without virus infection. It is possible.

Sulfurhodamine, which stains cells, may be used as Sulfurhodamine B, Sulfurhodamine 101, Sulfurhodamine G, or derivatives thereof. In order to stain the cells with sulfodadamine, the culture supernatant of the cells is removed and fixed with fixed solution such as Trichloroacetic acid (TCA), acetone, methanol, ethanol, glutaraldehyde or formalin. Sulfurhodamine should be dissolved completely in a solution such as distilled water. The cells are stained red for a period of time with sulforhodamine and the unstained sulfodadamine is removed by several washes. And basic solution, such as a Tris solution, is added, and it dissolves a sulfur of sulfur fully enough to measure the quantity of sulfur. The amount of sulfurhodamine can be analyzed by absorbance or fluorescence. In a specific embodiment of the present invention, absorbance was measured at 565 nm using Damine B (SRB) as sulfur.

In the present invention, "infection" refers to the introduction of the virus into the host cell, the cells that can be infected with the virus and look at the activity, eukaryotic cells, such as E. coli, prokaryotic cells, humans, cattle, pigs, chickens, shrimps, etc. It includes all the cells of origin. The cell may be a cell cultured directly from an organ or tissue or may be a cell line. Both adherent cells or suspension cells can be measured, and in the case of suspended cells, the culture medium is removed by centrifugation before fixing the cells.

Viruses whose activity can be measured by the methods of the present invention include all viruses that multiply in a soluble cycle. For example, Papovaviridae, Adenoviridae Herpesviridae, Foxviridae, Hepadnaviridae, Parvoviridae, Parvioviridae Reoviridae, Picornaviridae, Togaviridae, Cornaaviridae, Coryaviridae, Potyvirus, Timovirus, Tobamovirus, Comovirus ), Rhabdoviridae, Paramizoviridee (Paramyxoviridae), Orthomyxoviridae, Retroviridae, and the like, but are not limited thereto.

By measuring the virus activity using the sulfoda rhodamine, effective results can be obtained regardless of virus types such as corona virus, human rhinovirus, human influenza type A or B virus, swine rotavirus, bovine rotavirus, etc. I could confirm it. And because the 96-well can be used, large-capacity analysis is possible. At this time, the number of cells per well is not limited and the level of cells is quantified by objective absorbance or fluorescence, so there is no fear of misjudgment by subjective judgment. In addition, unlike the MTT analysis, since the morphology of the cells is not destroyed during the sulfodhodamine staining, the morphological characteristics of the virus infected cells can be observed under a microscope.

Through the activity measurement method, it is possible to quantitatively analyze the virus. In viral experiments, accurate quantification of viruses is required and the present invention provides a simple and reliable virus quantification method that improves on existing problems.

 It is also usefully applicable for the detection of antiviral agents.

In another embodiment, the present invention provides a method of culturing a cell to be infected with a virus; Infecting the cells with a virus; Culturing the cells with an antiviral candidate; Immobilizing the cells and staining with sulfurhodamine; Measuring the amount of sulfodadamine that binds to cellular proteins; A method for detecting an antiviral agent, the method comprising measuring the amount of sulfodadamine and comparing it to a control cell that is not infected with a virus.

As used herein, the term "candidate substance" means a substance suspected of having antiviral activity. Such candidate materials may include a single compound such as an organic or inorganic compound, a complex of a plurality of compounds, a high molecular compound such as proteins, carbohydrates and lipids, and the like.

After administration of the antiviral candidate, the extent to which virus proliferation is inhibited can be known from Equation 1. In a specific embodiment of the present invention, an effective antiviral agent that inhibits viral activity is compared by comparing the degree of cell breakage (BC) when the virus is infected with the degree of cell breakage (DC) when the virus is infected and the antiviral agent candidate is administered. Detected.

:

:

B: Absorbance in Virus Uninfected Cells

C: absorbance of virus in infected cells

D: absorbance in cells treated with both antiviral and virus

At this time, it can be said that it is an effective antiviral agent, when the virus growth inhibitory ability is suppressed 50% or more, preferably 70% or more, more preferably 90% or more within the effective amount range.

The term “effective amount” means a sufficient amount applicable to medical treatment or prevention in consideration of benefits and risk factors, and since an accurate result cannot be obtained below the effective amount range, the inhibitory capacity should be investigated within the effective amount range.

In another aspect, a virus quantitation kit and an antiviral agent search kit are provided.

The kit may include a virus, a host cell for culturing virus proliferation, a cell culture plate for attaching cells to the plate, and an agent for immobilizing the cell or cell protein on the plate, a washing solution, a bleaching solution, a sulforhodamine, and the like. .

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Materials and Virus Culture Methods

Reference Example 1 Preparation of SRB Staining Solution

0.4 g of sulfodadamine B purchased from Sigma was completely dissolved in distilled water in 1% (v / v) solution to prepare SRB staining solution.

Reference Example 2 Preparation of Bleaching Solution

Acetic acid was added to distilled water to 1% (v / v) to prepare a solution that was used as a decolorizing solution for sulfodadamine B staining.

Reference Example 3 Preparation of TCA Cell Fixed Solution

In order to fix the cultured cells on the plate TCA (Trichloroacetic acid) was dissolved in phosphate buffer solution to 10% to prepare a cell fixed solution.

Reference Example 4: Preparation of Acetone Cell Fixing Solution

In order to fix the cultured cells to the plate, acetone is added to distilled water to 70% (v / v) to prepare a cell fixed solution.

Reference Example 5 Preparation of Methanol or Ethanol Cell Fixing Solution (3)

Methanol or ethanol is added to distilled water so as to contain 70% (v / v) or more to prepare a cell fixed solution.

Reference Example 6: Preparation of glutaraldehyde or formalin cell fixation solution

A cell fixative is prepared by adding glutaraldehyde or formalin to 1% or 4% (v / v) in distilled water.

Reference Example 7 Preparation of Dye Dissolving Agent

Tris was dissolved in distilled water to 10 mM to prepare a dye solution.

Reference Example 8: Culture of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus stock solution, a type of corona virus stored in the laboratory of the present inventors, terminates the infection reaction and freezes and thaws when 90% of cells are crushed in the Vero cell culture plate due to viral infection. It was prepared by repeating twice. The activity of the virus stock solution is 100 pfu (plaque forming unit) / ml. Vero cells derived from monkey kidney tissue were used for the culture of swine pandemic diarrhea virus.

Reference Example 9: Culture of Human Influenza A Virus

Human influenza type A virus stored in the laboratory of the present inventors, the virus stock solution is finished when the virus was infected by 90% of cells in the MDCK cell culture plate due to the virus infection, and the virus stock solution was repeated twice by freezing and thawing. Prepared.

Reference Example 10 Culture of Human Influenza B Virus

Human influenza B virus stored in the laboratory of the present inventors, the virus stock solution when the virus was infected by 90% of the cells in the MDCK cell culture plate ended in the infection reaction and repeat the freeze and thaw process twice Prepared.

Reference Example 11 Culture of Porcine Rotavirus and Bovine Rotavirus

The porcine rotavirus and bovine rotavirus stocks stored in the laboratory of the present inventors finished the infection reaction when 90% of the cells were disrupted in the MA104 cell culture plate due to the virus infection, and the virus stock solution was repeated twice by freezing and thawing. Prepared.

Reference Example 12 Culture of Human Rhino Virus

The human rhinovirus stock solution stored in the laboratory of the present inventors finished the infection reaction when 90% of the cells were crushed in the HeLa cell culture plate due to the virus infection, and the virus stock solution was prepared by repeating the freezing and thawing process twice.

Example 2: Coronavirus Quantification Method

2 × 10 ⁴ Vero cells were placed in each well of 96 well plates and incubated for 24 hours. After 24 hours, the culture supernatant of each well was removed, and the porcine pandemic diarrhea virus stock solution in Reference Example 8 was serially diluted so that Vero cells were cultured at 25pfu, 5pfu, 1pfu, 0.2pfu, 0.04pfu, and 0.008pfu. After infection with the wells, the infection reaction was terminated after 72 hours, and the cells in the wells were fixed to the plate with the fixed solution prepared in Reference Example 4. After the plate was completely dried, the cell protein was stained at room temperature for 30 minutes with the SRB staining solution prepared in Reference Example 1. SRB stains that did not bind to the cell protein were completely removed by washing 5 times with 1% (v / v) acetic acid and dried again. In this process, as the virus activity was lowered, the number of cells remaining in the wells was increased (FIG. 1). The morphology was observed under a microscope, and the morphology of the cells was photographed with a camera attached to the microscope (FIG. 2). 100 μl of 10 mM Tris solution (pH 10.5) was added to each well to fully dissolve the staining agent bound to the cells, and then the absorbance was measured at 565 nm. The absorbance of the wells was calculated by comparison and presented in FIG. 3.

Example 3: Method of Quantifying Human Influenza A Virus

1 × 10 ⁴ MDCK cells were placed in each well of a 96 well plate and incubated for 24 hours. After 24 hours, the culture supernatant of each well was removed, and the human influenza A virus stock solution in Reference Example 9 was serially diluted to infect each well in which the MDCK cells were cultured. The cells in the wells were fixed to the plate with the prepared fixed solution. After the plate was completely dried, the cell protein was stained at room temperature for 30 minutes with the SRB staining solution prepared in Reference Example 1. SRB stains that did not bind to the cell protein were completely removed by washing 5 times with 1% (v / v) acetic acid and dried again. In this process, the number of cells remaining in the wells increased as the virus activity decreased. 100 μl of 10 mM Tris solution (pH 10.5) was added to each well to sufficiently dissolve the dye bound to the cells, and then absorb the absorbance at 565 nm. The absorbances of the wells infected with the virus were compared and calculated as percentages based on the absorbance average of the measured and non-virus infected wells at 100%, and are shown in FIG. 4.

Example 4 Human Influenza B Virus Quantitative Methods

1 × 10 ⁴ MDCK cells were placed in each well of a 96 well plate and incubated for 24 hours. After 24 hours, the culture supernatant of each well was removed, and the human influenza B virus stock solution in Reference Example 10 was serially diluted to infect each well in which the MDCK cells were cultured. The cells in the wells were fixed to the plate with the prepared fixed solution. After the plate was completely dried, the cell protein was stained at room temperature for 30 minutes with the SRB staining solution prepared in Reference Example 1. SRB stains that did not bind to cellular proteins were washed five times with 1% (v / v) acetic acid and completely removed and dried again. In this process, the number of cells remaining in the wells increased as the virus activity decreased. 100 μl of 10 mM Tris solution (pH 10.5) was added to each well to sufficiently dissolve the dye bound to the cells, and then absorb the absorbance at 565 nm. The absorbance of the virus-infected wells was calculated by comparison as a percentage, and the absorbance average of the wells which did not infect the virus was 100%, and is shown in FIG. 5.

Example 5 Methods for Quantifying Porcine Rotavirus or Bovine Rotavirus

2 × 10 ⁴ MA104 cells were placed in each well of a 96 well plate and incubated for 24 hours. After 24 hours, the culture supernatant of each well was removed and serial dilutions of porcine rotavirus and bovine rotavirus stock solution in Reference Example 11 were performed to infect each well in which MDCK cells were cultured. The cells in the wells were fixed to the plate with the fixed solution prepared in step 4. After the plate was completely dried, the cell protein was stained at room temperature for 30 minutes with the SRB staining solution prepared in Reference Example 1. SRB stains that did not bind to the cell protein were completely removed by washing 5 times with 1% (v / v) acetic acid and dried again. In this process, the number of cells remaining in the well increased as the virus activity decreased. To each well, 100 µl of 10 mM Tris solution (pH 10.5) was added to dissolve the dye bound to the cells, and then absorbed at 565 nm. The absorbance of the virus-infected wells was calculated and compared in terms of percentages, with the average absorbance of the wells measured and not infected with viruses at 100%.

Example 6: Antiviral activity screening method using the virus quantification method of the present invention

2 × 10 ⁴ Vero cells were placed in each well of a 96 well plate and incubated for 24 hours. After 24 hours, the culture supernatant of each well was removed, and the porcine epidemic diarrhea virus stock solution in Reference Example 8 was diluted and infected with each well in which Vero cells were cultured at an activity concentration of 0.5 pfu, 0.04 pfu, and 0.008 pfu, and then sigma Ribavirin, acyclovir, and lamivudine, purchased from GlaxoWelcomeCom, were administered to virus-infected wells and virus-free wells at concentrations of 100, 10, 1, and 0.1 µg / ml, respectively. After 72 hours, the infection reaction was terminated, and the cells in the wells were fixed to the plate with the fixed solution prepared in Reference Example 4. After the plate was completely dried, the cell protein was stained at room temperature for 30 minutes with the SRB staining solution prepared in Reference Example 1. SRB stains that did not bind to the cell protein were completely removed by washing 5 times with 1% (v / v) acetic acid and dried again. In this process, it was confirmed that the antiviral activity was different according to the characteristics of the drug, the shape was observed under a microscope, and the shape of the cells was photographed with a camera attached to the microscope (Fig. 7). 100 μl of 10 mM Tris solution (pH 10.5) was added to each well to sufficiently dissolve the dye bound to the cells, and the absorbance was measured at 565 nm. Determination of antiviral activity is expressed by the absorbance (B) of the wells not infected with the virus, the absorbance (C) of the wells infected with the virus, and the absorbance (D) of the wells administered with the antiviral agent and the virus. Calculated according to the results according to the concentration of each drug is shown in FIG.

Equation 1

In FIG. 7, the antiviral activity of each drug was observed at a concentration of 10 μg / ml. Only ribavirin showed the inhibitory effect of viral activity according to the change of concentration. Acyclovir and lamivudine did not show antiviral activity without any difference in concentration.

The virus activity measuring method using the sulfoda rhodamine of the present invention is quantified by an objective absorbance or fluorescence value, so that there is no fear of misjudgment by subjective judgment, the measurement result is accurate and reliable, large-capacity analysis is possible, and the procedure is simple. In addition, since it is inexpensive, when using the assay of this invention, antiviral agent can be detected usefully.

Claims (6)

Culturing the cells to be infected with the virus; Infecting the cells with a virus; Inactivating and staining the cells with sulfurforhodamine; Measuring the amount of sulfodadamine that binds the cellular protein and comparing it to a control cell that is not infected with the virus. The method of claim 1, wherein the sulfodadamine is sulfodadamine B, sulfodadamine 101, sulfodadamine G, or a derivative thereof. The method of claim 1, which is measured using absorbance or fluorescence. Culturing the cells to be infected with the virus; Infecting the cells with a virus; Culturing the cells with an antiviral candidate; Inactivating and staining the cells with sulfurforhodamine; Measuring the amount of sulfodadamine that binds to cellular proteins; A method for detecting an antiviral agent, the method comprising measuring the amount of sulfodadamine and comparing it to a control cell that is not virus infected. The method of claim 4, wherein the sulfodadamine is sulfodadamine B, sulfodadamine 101, sulfodadamine G, or a derivative thereof. The method of claim 4, which is measured using absorbance or fluorescence.

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