Method for efficiently screening and evaluating anti-plant-virus compounds based on insect cells

Abstract

In order to solve the problem that an efficient and stable anti-plant-virus compound screening and evaluating technology is lacked, the invention provides an efficient anti-plant-virus compound screening and evaluating method based on insect cells. An insect cell line Sf9JSN106 which is obtained through domestication and can maintain infection and replication of plant viruses (representative species: rice stripe viruses) is used for rapidly screening and evaluating the antiviral effect of a compound. Evaluation is carried out from two aspects: 1) evaluating the inhibition effect of the compound on virus replication, wherein the process comprises cell inoculation, virus cell infection, medicament treatment and virus detection; and 2) evaluating the inhibition effect of the compound on virion infection activity, wherein the process comprises cell inoculation, virus treatment by a medicament, cell infection by the virus and virus detection. The method is completed by using a cell culture plate, various compounds can be evaluated at a time, the whole process does not exceed 60 hours, and the method has the characteristics of simplicity, convenience, rapidness and high efficiency, and can be applied to screening evaluation of plant virus resistant compounds and antiviral mechanism research.

Description

Technical Field

[0001] The present invention relates to a method for rapidly and efficiently evaluating the anti - plant virus effect of compounds using insect cells, and belongs to the field of plant protection in agricultural science. Background Art

[0002] Plant virus diseases are numerous in variety, wide in host range, great in harm, and difficult to control. They are the second - largest plant diseases after fungal diseases. More than 1300 plant viruses have been reported, damaging more than 1000 kinds of plants, resulting in a serious decline in crop yield and quality. According to incomplete statistics, the annual economic loss of crops caused by plant virus diseases globally is as high as $60 billion, posing a great threat to the safe production of crops. The adjustment of planting structure and climate change have led to the increasingly common occurrence of plant virus diseases in China, showing the characteristics of suddenness, explosiveness, and recurrence, seriously affecting food production and the safety of the supply chain of important agricultural products. At present, the prevention and control of plant virus diseases in production mainly rely on agricultural measures and disease - resistant varieties. However, there are still prominent problems such as the lack of disease - resistant varieties and the degradation of resistance in the prevention and control of many virus diseases. Antiviral agents are still the most direct and effective means for preventing and controlling plant virus diseases. Currently, plant virus disease control agents are extremely scarce. The proportion of registered antiviral agent products among all registered pesticide products is less than 0.5%. Although there are some antiviral agent products on the market, such as moroxydine hydrochloride, oligosaccharins, dufulin, and xinjunamine, plant viruses are complex in variety and diverse in infecting and harming crops. The existing agents play a very limited role in preventing and controlling crop virus diseases. There are no registered antiviral agent products for many newly emerging or explosive virus diseases, resulting in the dilemma of having no available drugs for the prevention and control of many virus diseases in production. Therefore, it is urgent to accelerate the research and development of antiviral agents.

[0003] Currently, there are two main bottlenecks restricting the development of anti - plant virus compounds: First, the research and development of antiviral compounds itself belongs to the research and development of new pesticides, which is a systematic project involving multiple disciplines and covering multiple fields, with a long cycle, high threshold, large investment, and high risk. Second, plant viruses are obligate parasitic organisms with a large number of species, and their evaluation is relatively complex. The traditional method of relying on directly spraying the agent on diseased plants for evaluation has a long cycle, huge workload, and unstable results. Currently, many domestic units and enterprises have carried out research on related antiviral lead compounds, providing primary samples for the creation of new antiviral agent products. However, the current lack of a systematic, stable, and perfect antiviral agent screening system severely restricts the research and development of new antiviral agents. Therefore, establishing an efficient and stable high - throughput screening and evaluation technology method for antiviral agents has an important promoting effect on the creation of new antiviral agents.

[0004] The mechanisms of action of antiviral compounds can generally be divided into two categories. One is to directly target viruses and their factors, inhibiting virus replication, proliferation, and infection in plants. The other is to induce the plant's disease resistance signaling pathway, activate the plant's immune defense system, and thus enhance the plant's resistance level to viral diseases. In the traditional method of evaluating antiviral compounds by spraying infected plants with agents, in addition to the long cycle and unstable results, it is also impossible to determine the mechanism of action of antiviral compounds based on the symptoms of plants in the experiment, which hinders the subsequent development and application of compounds. The use of cell lines can achieve rapid evaluation of the effects of agents. Currently, plant cells (protoplasts) grow slowly and are difficult to subculture and maintain, making them unsuitable for agent evaluation. Most plant viruses are transmitted by insect vectors, which are also the insect hosts of the viruses, making it possible to screen and evaluate the anti-plant virus effects of compounds using insect cells.

[0005] This method uses an insect cell line obtained by domestication that can maintain the infection and replication of plant viruses to achieve rapid screening and evaluation of the anti-plant virus effects of compounds. Since the evaluation is carried out based on insect cells, it avoids the plant's immune defense system and can specifically evaluate antiviral compounds targeting viruses. On the premise of ensuring accuracy and reliability, the characteristics of rapid cell culture make this method have simplified steps, high throughput, and save evaluation costs and time. This method innovates the technical system for screening and evaluating anti-plant virus compounds, helps to solve the problem of difficult evaluation in the research and development process of new, highly efficient, and safe antiviral agents, and is of great significance for improving the prevention and control level of plant virus diseases in China. Summary of the Invention

[0006] In view of the above research background, the present invention has developed a high-efficiency screening and evaluation method for anti-plant virus compounds based on insect cells. Using an insect cell line Sf9 obtained by domestication that can maintain the infection and replication of plant viruses (representative species: Rice stripe virus, RSV) JSN106 , after virus infection and compound agent treatment, the anti-plant virus effects of the compounds are evaluated by detecting and analyzing the virus content in the cells. The evaluation method includes two levels: 1) Evaluation of the inhibitory effect of the compound on virus replication: Insect cell line Sf9 is inoculated with RSV JSN106 . After 4 hours, a certain concentration of candidate compound (within the safe concentration range of the agent) is added to the cell culture medium. After 24 hours of treatment, cell samples are collected for virus quantification detection. By comparing with the virus content in the cells of the blank control group, the inhibitory effect of the candidate compound on virus replication can be analyzed; 2) Evaluation of the inhibitory effect of the compound on the infectivity of virus particles: A certain concentration of candidate compound (within the safe concentration range of the agent) is incubated with RSV virus solution at room temperature for 2 hours, and then inoculated into insect cell line Sf9 JSN106, collect cell samples after 3 hours for virus quantification detection. By comparing with the virus content in the cells of the blank control group, the inhibitory effect of the candidate compound on the infectivity of virus particles, that is, the inactivation effect of the compound on virus particles, can be analyzed ( Figure 1 ). The present invention provides a method for screening and evaluating the anti-plant virus effect of compounds based on insect cells. Under the condition of normal maintenance of the cell line, the whole process only includes steps such as cell inoculation, virus infection, agent treatment, and virus detection, and the time can be controlled within 60 hours, which reflects the characteristics of simplicity and rapidity. The evaluation operation is completed using a 24-well or 48-well cell culture plate, and multiple compounds can be evaluated at one time, with the advantages of high efficiency and high throughput. It is a simple, rapid, efficient, and economical method for screening and evaluating anti-plant virus compounds, filling the technical gap of rapid evaluation using insect cells in the evaluation of anti-plant virus compounds, and can be applied to the screening of anti-plant virus compounds, effect evaluation, and research on the antiviral mechanism of compounds, promoting the creation and research and development of new antiviral agents in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 : Schematic diagram of the process for highly efficient screening and evaluation of anti-plant virus compounds based on insect cells.

[0008] Figure 2 : Accumulation amount of virus in cells treated with different concentrations of agent (Lentinan) after Rice stripe virus (RSV) infects insect cells.

[0009] Figure 3 : Inhibition rate (A) and half-inhibitory concentration IC 50 (B) of virus after treatment with different concentrations of agent (Lentinan) after RSV infects insect cells.

[0010] Figure 4 : Detection of virus infection amount in cells after treating RSV with agent (Lentinan). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0011] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0012] The experimental methods used in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0013] The insect cell line Sf9 used in the embodiments of the present invention JSN106 was obtained by domestication in the laboratory by the inventor. The plant virus used is Rice stripe virus (RSV), which is provided by the inventor's laboratory. The rest of the materials, reagents, etc. can all be obtained from commercial channels.

[0014] In the embodiments of the present invention, lentinan (LNT) is selected as the candidate compound for evaluation to illustrate the present invention in detail.

[0015] Example 1: Evaluation of the inhibitory effect of the compound on virus replication

[0016] Using lentinan as the candidate compound, its inhibitory effect on the replication and proliferation of RSV was evaluated.

[0017] 1. Insect cells and virus

[0018] The insect cell line Sf9 used JSN106 Is domesticated from Sf9 insect cells, can be efficiently infected by RSV, and can maintain the replication of RSV in cells. The cryopreserved Sf9 JSN106 Cells were thawed and resuscitated, then placed in a cell incubator at 28°C for standby culture, and passaged once every 7 days.

[0019] The RSV used in the experiment was stored in the laboratory. The viruliferous vector Laodelphax striatellus was transferred to rice seedlings (variety: Wuyujing 3) at a ratio of 1 Laodelphax striatellus per seedling for virus transmission. After 1 day, the seedlings were transferred to the field for growth. After 30 days, the disease incidence of rice was observed, and diseased leaves were collected for inoculating cells or stored at -70°C.

[0020] 2. Cell inoculation

[0021] After microscopic examination, the cells cultured to a full and good morphology and in the vigorous growth period were suspended with fresh insect cell medium (Gibco Sf-900Ⅱ SFM). After measuring the cell concentration, the medium was supplemented to adjust the cell concentration to (0.8 - 1)×10 6 cells / mL, and inoculated into the wells of a 24-well cell culture plate at 0.5 mL / well, and placed in a cell incubator at 28°C for 1 h to allow the cells to fully adhere to the bottom of the well, and then virus infection could be carried out.

[0022] 3. Virus infection method

[0023] Take 1 g of fresh or frozen diseased leaves, cut them into small pieces, grind them into powder in liquid nitrogen, add an appropriate amount (5 - 6 mL) of 0.1M PBS buffer and homogenize for 5 min, transfer to a 15 mL centrifuge tube, centrifuge at 5000 rpm for 5 min, take the supernatant and sterilize it with a 0.22 μm bacterial filter, and thus complete the preparation of the RSV virus solution. Add the virus solution to the wells at 90 μL / well, gently shake the culture plate to mix evenly, and place it in a cell incubator at 28°C for incubation for 4 h.

[0024] 4. Medicament treatment

[0025] After incubating the virus solution with the cells for 4 h, aspirate and remove the culture medium in the well of the plate, wash the surface of the cell layer once with PBS buffer, add the culture medium containing lentinan at gradient concentrations (12.5, 25, 50, 100, 200 μg / mL) to the well of the cell plate, and only add the culture medium to the control group. Incubate at 28 °C for 24 h. Set 3 replicates of cell plate wells for each treatment group.

[0026] 5. Virus detection and inhibition effect analysis

[0027] Suspend and collect the cells in the well plate, wash the cells once with PBS buffer, after centrifugal enrichment, fully lyse the cells by the method of repeated freezing and thawing. Extract the total RNA of the cells by the Trizol method, reverse transcribe to synthesize the first-strand cDNA, and then perform fluorescence quantitative PCR to detect the accumulation of RSV in the cells. Use the RSV CP gene as the detection target and the sf9 cell β-Actin gene as the internal reference. The primers used are as follows: qCP-F: 5’-TGCAGAAGGCAATCAATGACAT-3’, qCP-R: 5’-TGTCACCACC TTTGTCCTTCAA-3’, qActin-F: AAGGACCTGTACGCCAACAC-3’, qActin-R: 5’-ACATCTG CTGGAAGGTGGA-3’. The reaction system is 20 μL: 2×SYBR Green PCR Master Mix 10 μL, cDNA 1 μL, upstream primer 1 μL, downstream primer 1 μL, ddH 2 O 7 μL. The reaction conditions are pre-denaturation at 95 °C for 30 sec; denaturation at 95 °C for 5 sec, annealing at 60 °C for 15 sec, 40 cycles; melting curve analysis, from 60 °C to 95 °C, increasing at 0.1 °C / sec. After obtaining the Ct values of each sample, use the 2 -ΔΔCt method to calculate the relative level of the CP gene in the samples of each treatment group. The results show ( Figure 2 ), after treatment with lentinan, the accumulation of the virus in the cells decreased significantly. Compared with the control group, the relative contents of the virus in the cells of the 25, 50, 100, and 200 μg / mL treatment groups decreased by 32.67%, 48.77%, 60.27%, and 68.70% respectively, and the relative content of the virus in the cells of the 12.5 μg / mL treatment group was similar to that of the control group, with no significant difference.

[0028] Calculate the inhibition rate of the medicament on the virus and the half-inhibitory concentration IC 50。Inhibition rate = (Relative virus expression in the control group - Relative virus expression in the treatment group) / Relative virus expression in the control group × 100%. The inhibition rates of lentinan (LNT) at 5 experimental concentrations (12.5, 25, 50, 100, 200 μg / mL) against RSV in cells were 8.17%, 32.67%, 48.77%, 60.27% and 68.70% respectively( Figure 3 ). Half-inhibitory concentration IC 50 Calculation: Take the logarithm of the LNT concentration to the base 10, i.e., lg[(LNT)(μg / mL)] as the abscissa, and the inhibition rate as the ordinate to plot a graph, and obtain the linear regression equation y = 49.386x - 40.192 (R 2 = 0.957). When the inhibition rate is 50%, the IC 50 of LNT is obtained as 66.99 μg / mL( Figure 3 ).

[0029] Based on the above results, the conclusion is drawn that lentinan has an inhibitory effect on the replication of plant viruses, and its half-inhibitory concentration IC 50 against RSV is 66.99 μg / mL.

[0030] Example 2: Evaluation of the inhibitory effect of a compound on the infectivity of virus particles

[0031] Lentinan was used as a candidate compound to evaluate its inhibitory effect on the infectivity of RSV particles.

[0032] 1. Insect cells and virus

[0033] The insect cells and virus used were the same as in Example 1.

[0034] 2. Cell seeding

[0035] The cell seeding method was the same as in Example 1.

[0036] 3. Agent treatment and virus infection

[0037] The preparation of the RSV virus solution was the same as in Example 1. The filtered and sterilized lentinan solution was added to the virus solution to make its final concentrations 100 μg / mL and 200 μg / mL respectively. The control group was the addition of an equal volume of PBS buffer to the virus solution. The compound and the virus were incubated at room temperature for 2 h. Each treatment group was set with 3 replicates of cell plate wells. The virus incubation solution was added to the plate wells at 90 μL / well, and the culture plate was gently shaken to mix evenly and incubated in a 28°C cell culture incubator for 3 h to complete virus infection.

[0038] 4. Virus detection and analysis of inhibitory effect

[0039] Suspend and collect the cells, wash the cells 3 times with PBS buffer to remove extracellular viruses, detect the virus content in the cells. At this time, the virus has just completed the process of infecting the cells, and the virus content in the cells can reflect the amount of virus invasion, that is, it can reflect the infectivity of the virus. The virus detection method is the same as that in Example 1. The results show that ( Figure 4 ), after treating the virus with lentinan, the virus content in the cells was significantly lower than that in the control group. The relative virus contents in the cells of the 100 and 200 μg / mL treatment groups decreased by 44.09% and 59.76% respectively, indicating that the infectivity of RSV particles decreased significantly after treatment with lentinan.

[0040] Based on the above results, it can be concluded that lentinan has an inhibitory effect on the infectivity of plant virus particles, that is, lentinan has a passivating effect on plant virus particles.

[0041] The above embodiments do not limit the present invention in any form.

Claims

1. A highly efficient screening and evaluation method for anti-plant virus compounds based on insect cells, characterized in that: Using an insect cell line that can sustain infection and replication of plant viruses (representative species: rice stripe virus), after virus infection and compound treatment, the anti-plant virus effect of the compound is quickly screened and evaluated by detecting the intracellular virus content.

2. The method for screening and evaluating anti-plant virus compounds according to claim 1, characterized in that: The evaluation method includes the following two levels: 1) Evaluation of the inhibitory effect of the compound on viral replication, the process includes cell inoculation, virus infection of cells, drug treatment of cells and virus detection and analysis; 2) Evaluation of the inhibitory effect of the compound on the infectious activity of viral particles, the process includes cell inoculation, drug treatment of viruses, virus infection of cells and virus detection and analysis.

3. A method for screening and evaluating anti-plant virus compounds according to claims 1 to 2, characterized in that: The insect cell is an insect cell line that can maintain the infection and replication of plant viruses.

4. The feature according to claim 3, characterized in that: The insect cell line capable of maintaining plant virus infection and replication is an insect cell line Sf9 capable of maintaining rice stripe virus infection and replication obtained by domesticating Sf9 cells. JSN106 .

5. A method for screening and evaluating anti-plant virus compounds according to claims 1 to 3, characterized in that: The plant viruses include, but are not limited to, rice stripe virus, and also include other plant viruses that can infect and replicate in insect cells.

6. A method for screening and evaluating anti-plant virus compounds according to claims 1 to 3, characterized in that: The compounds should include all compounds that are safe and non-toxic to insect cells, that is, all compounds that are safe to insect cells can be evaluated for antiviral effects using this method.

7. A method for screening and evaluating anti-plant virus compounds according to claims 1 to 3, characterized in that: When screening and evaluating compounds, the concentration of the agent used should be within the safe concentration range of insect cells.

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