Method for rapidly determining virus replication cycle

Abstract

The invention belongs to the technical field of biological medicine. More specifically, the invention relates to a method for rapidly determining the virus replication period. Through high-frequency sampling after infection and combination of virus specific fluorescent antibody labeling and continuous observation in the same visual field, the invention provides a brand-new rapid determination method for the virus replication period. The method does not need to depend on an animal model and is completely completed in an in-vitro cell system, so that the operation cost is remarkably reduced; the shortest total experiment time can be controlled within 24 hours, and the research and development efficiency is greatly improved. The determination method provided by the invention has high specificity and sensitivity, is simple and convenient to operate, low in equipment threshold and suitable for various viruses, provides a brand new method for determining the virus replication period with speed, precision and feasibility for basic research of virology and research and development of antiviral products, and has good application prospects and application values.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a method for rapidly determining the viral replication cycle. Background Technology

[0002] Viruses reproduce by replication. Under the regulation of their genes, viruses synthesize their nucleic acid and protein components, which are then assembled into infectious viral particles in the cell nucleus or cytoplasm. These particles are then released outside the cell in various ways to infect other cells, initiating another infection cycle. This process is called replication, and the entire process is called the replication cycle. The viral replication cycle refers to the entire process from viral adsorption to host cells, including penetration, uncoating, biosynthesis, assembly, and the release of progeny viral particles. It is a key biological parameter that determines viral infection dynamics, pathogenicity, and the intervention window. Accurate determination of the replication cycle plays an irreplaceable role in understanding viral pathogenic mechanisms, evaluating the effectiveness of antiviral drug targets, and optimizing vaccine production processes.

[0003] Currently, there is no standard method for detecting the viral replication cycle. Detection is primarily achieved through a combination of animal methods, molecular biology, microscopic imaging, and biochemistry, including in vivo animal inoculation, qPCR, Western blotting, ELISA, and flow cytometry. These methods have the following drawbacks: in vivo animal inoculation requires a large number of animals, animals may not be sensitive (e.g., influenza virus may not cause animal death), there is significant individual variability in animals, and the experimental cycle is long; qPCR cannot distinguish between live and inactivated viruses; Western blotting is cumbersome and has low throughput; ELISA generally has low sensitivity and reproducibility; and flow cytometry equipment is expensive, operation is complex, and it cannot detect non-enveloped viruses, making it difficult to implement in routine laboratories.

[0004] Therefore, there is an urgent need to develop a new rapid assay method that is easy to operate, has a short cycle (≤24 h), specifically distinguishes the infection process of live viruses, does not require animals, and is compatible with conventional cell and fluorescence equipment, in order to fill the technological gap and provide reliable and standardized tool support for virological mechanism research, high-throughput screening of antiviral drugs, and optimization of vaccine processes. Summary of the Invention

[0005] The present invention aims to overcome the defects and deficiencies in the prior art and provide a simple and short method for determining the viral replication cycle.

[0006] The purpose of this invention is to provide a method for determining the viral replication cycle and its application.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: Current research, titled "Observation of the Replication Cycle of Rabies Virus CTN-1 Strain in Vero Cells," indicates that the viral replication cycle of rabies virus in Vero cells is approximately 16 hours. The method provided in this invention determined the replication cycle of rabies virus PM strain in BHK-21 cells to be 16 hours, which is highly consistent with results obtained in existing technologies, verifying its accuracy and reliability.

[0008] Therefore, this invention provides a method for determining the viral replication cycle, which involves infecting cells with the virus to be tested, continuously monitoring cell samples, and observing the number of infected cells; recording the time point at which infected cells are first observed as T1, and the corresponding proportion of infected cells as A1; and recording the corresponding time as T until the proportion of infected cells is greater than or equal to k·A1. n Then the virus replication cycle T = T n T1; k is a real number greater than or equal to 2.

[0009] Specifically, the infected cell ratio is the proportion of infected cells to the total number of cells.

[0010] Preferably, the time interval for continuous monitoring is 1 to 6 hours.

[0011] More preferably, the time interval is 1 to 3 hours.

[0012] More preferably, the time interval is 2 hours.

[0013] Optionally, k can be any integer among 2, 3, 4, and 5.

[0014] Preferably, the multiplicity of infection (MOI) of the virus is 0.001 to 0.005 (preferably 0.002). Maintaining an MOI of 0.001 to 0.005 ensures an average initial number of infected cells of 10 to 30 per field of view. The MOI refers to the ratio of virus to cells at the time of infection.

[0015] Optionally, the virus is a virus that can be recognized by a specific fluorescent antibody.

[0016] Optionally, the virus is a virus from the Orthomyxoviridae family, Paramyxoviridae family, Herpesviridae family, Coronaviridae family, Retroviridae family, or Rhabdoviridae family.

[0017] Optionally, the virus may be rabies virus, influenza virus, COVID-19 virus, or HIV virus.

[0018] The viruses of the Orthomyxoviridae family include influenza A / B viruses, etc.; the viruses of the Paramyxoviridae family include measles virus, mumps virus, respiratory syncytial virus, etc.; the viruses of the Herpesviridae family include herpes simplex virus, varicella-zoster virus, etc.; the viruses of the Coronaviridae family include SARS-CoV-2, SARS virus, etc.; the viruses of the Retroviridae family include HIV virus, human T-cell leukemia virus type I, etc.; and the viruses of the Rhabdoviridae family include rabies virus, vesicular stomatitis virus, etc.

[0019] As an alternative implementation, the cells in the above method include at least one of BHK-21 cells, BSR cells, Vero cells, MDCK cells, or HEK293T cells.

[0020] As an alternative implementation, the above method involves labeling cell samples with virus-specific fluorescent antibodies for quantitative observation.

[0021] Optionally, the specific fluorescent antibody is a monoclonal antibody or polyclonal antibody that specifically binds to viral nucleoproteins.

[0022] Optionally, the specific fluorescent antibody is any one of FITC, TRITC, Cy3, and Cy5.

[0023] Specifically, the observation is performed under the same magnification and field of view conditions. Preferably, the microscope has a magnification of 50x (more preferably 25-75x).

[0024] Specifically, the same field of view refers to selecting a fixed position in the central region of the observed object.

[0025] In this embodiment of the invention, the object of observation is a microplate. Optionally, the stage coordinates are kept constant, with the center of the cross intersection of the bottom central region of the microplate holes as a reference, to ensure a consistent field of view.

[0026] As an alternative implementation scheme, the above method for determining the viral replication cycle is as follows: Infect cells with the virus to be tested, monitor cell samples at fixed time intervals, label samples at each time point with virus-specific fluorescent antibodies, observe and count the number of infected cells; record the time point at which infected cells are first observed as T1, and the corresponding number of infected cells as A1; continue to observe the number of infected cells at subsequent time points, and when the number of infected cells A1 first appears... n Satisfy A n When k ≥ A1, record the corresponding time as T. n Then the virus replication cycle T = T n T1; k is a real number greater than or equal to 2; the time interval is 1 to 6 hours.

[0027] The present invention has the following beneficial effects: This invention provides a rapid method for determining the viral replication cycle based on dynamic fluorescent focus counting. By frequently sampling after infection, combined with virus-specific fluorescent antibody labeling and continuous observation in the same field of view, a novel and rapid method for determining the viral replication cycle is provided. This method does not rely on animal models and is performed entirely in an in vitro cell system, significantly reducing operating costs; the total experimental time can be controlled to a minimum of 24 hours, greatly improving research and development efficiency.

[0028] The assay method of this invention possesses high specificity and sensitivity. It employs fluorescently labeled antibodies targeting viral structural proteins (such as nucleoproteins or glycoproteins) to specifically identify live cells that have undergone effective infection and express viral proteins. Furthermore, the assay method of this invention is simple to operate and requires minimal equipment, needing only conventional cell culture facilities, a fluorescence microscope, and basic antibody reagents. It is applicable to various viruses that can be recognized by specific antibodies, such as rabies virus, and has good universality. Simultaneous detection of multiple viruses or under multiple conditions can be achieved through parallel multi-well plate design, making it suitable for initial screening and mechanism of action studies of antiviral drugs. The assay method of this invention determined the replication cycle of rabies virus PM strain in BHK-21 cells and BSR cells to be 16 hours, which is highly consistent with results obtained in existing technologies, verifying its accuracy and reliability.

[0029] In summary, this invention provides a method for rapidly determining the viral replication cycle, offering a new tool that combines speed, accuracy, and feasibility for basic virology research and the development of antiviral products, and has great application prospects and value. Attached Figure Description

[0030] Figure 1 The results are observed by fluorescence microscopy of rabies virus at an incubation time of 6 hours.

[0031] Figure 2 The results are observed by fluorescence microscopy of rabies virus at an incubation time of 8 hours.

[0032] Figure 3 The results are observed by fluorescence microscopy of rabies virus at an incubation time of 22 h.

[0033] Figure 4 The results are observed by fluorescence microscopy of rabies virus at an incubation time of 24 h. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] In the following examples, the rabies virus is the PM strain of rabies virus.

[0037] FITC rabies virus fluorescent antibody, manufacturer: Merck, batch number: 3568436.

[0038] In the following examples, the fluorescence microscope (brand: Leica, model: DMI3000B) was adjusted as follows: the fluorescence intensity knob was set to the maximum setting of 100%, and the fluorescence filter turntable was set to the second setting, L5. This is a combination system of blue light excitation / green light emission fluorescence filters. The excitation filter is BP 480 / 40nm, the emission filter is BP527 / 30nm, and the dichroic mirror is 505nm. This system is suitable for FITC fluorescent dyes.

[0039] Example 1 I. Sample Dilution Take the frozen rabies virus sample, thaw it with running water, dilute the virus to a limited extent with DMEM culture medium (containing 10% serum), and then mix thoroughly.

[0040] II. Measurement Method 1. Adding samples Rabies virus samples were added to the reaction wells at a rate of 20 viruses per well, 20 μl / well.

[0041] 2. Add cells Add 20 μL of a 5×10⁻⁶ concentration to each well. 5 Hamster kidney fibroblast (BHK-21) cell suspension was prepared by adding 40 μL of DMEM culture medium (containing 10% serum) to each well, resulting in a total of 80 μL per well. The microplates were then cultured at 37°C and 5% CO2 for 0–24 h. 13 independent parallel plates were set up at 2-hour intervals for dynamic observation.

[0042] 3. Fix After the microplate culture at each time point was completed, the supernatant was discarded, and the microplate was washed once with 80 μL PBS and dried. 50 μL of 80% acetone pre-cooled to 4℃ was added to each well, and the microplate was fixed at 4℃ for 30 min (or at -30℃ for 10 min).

[0043] 4. Antibody incubation Discard the acetone and allow it to evaporate and dry. Add 20 μl of antibody working solution (FITC rabies virus fluorescent antibody) to each well and incubate at 37°C for 30 minutes.

[0044] 5. Observation Discard the antibody solution, wash the plate three times with PBS, spin dry, add 20 μl of 80% glycerol to each well, and observe under a fluorescence microscope.

[0045] 6. Observation and Result Judgment An inverted fluorescence microscope was used to continuously observe samples at each time point with the same field of view, with a 2-hour interval between each time point. Under the inverted fluorescence microscope, an overview of the entire well field of view was initially obtained at 50× total magnification (5× objective lens, 10× eyepiece lens) for counting; during imaging, the magnification was switched to 100× total magnification (10× objective lens, 10× eyepiece lens) to ensure that the cell imaging effect met expectations.

[0046] The determination and calculation methods for the virus replication cycle are as follows: (1) Define the incubation time point at which the first typical fluorescent spot (i.e., the fluorescent spot formed in the cell after the virus infects the cell, representing the number of cells infected by the virus) appears as T1, and the corresponding number of fluorescent spots is A1 (based on the number of cells in which fluorescent spots are observed, reflecting the initial number of infected cells). (2) Subsequent time points are denoted as T. i (i = 2, 3, …, n), corresponding to A fluorophores. i ; (3) When A is satisfied for the first time n When k ≥ k·A1 (k ≥ 2, in this embodiment k = 3), the corresponding time is T. n Then the virus replication cycle T = T n – T1.

[0047] Infected cell ratio = Number of virus-infected cells / Total number of cells (1×10⁻⁶) 4 indivual).

[0048] III. Calculation Results The results of observing the number of fluorescent foci at different incubation time points are shown in Table 1. The results indicate that: No fluorescence signal was observed at any time point from 0 to 6 hours. Figure 1 Typical fluorescent foci were first observed at T1 = 8 h, with a count of 20 foci (A1), consistent with the inoculated viral load. Figure 2 Subsequently, at T8 = 22 h, the number of fluorophores A8 was 28 (). Figure 3 ); T9 = 24 h, the number of fluorescent foci A9 is more than 60, satisfying the condition A9≥3·A1 ( Figure 4 Therefore, the replication period T = 24 h. 8 h = 16 h.

[0049] The results are highly consistent with the literature reports on the replication cycle (16-20 h) of rabies virus in BHK-21 or Vero cells, verifying the accuracy and reliability of this method.

[0050] Table 1. Observation results of the number of fluorescent foci at different incubation time points

[0051] Note: All observations at the same time point were conducted under the same field of view and parameters to ensure data comparability; the experiment was repeated 3 times and the results were consistent.

[0052] Example 2 I. Sample Dilution Take the frozen rabies virus sample, thaw it with running water, dilute the virus to a limited extent with DMEM culture medium (containing 10% serum), and then mix thoroughly.

[0053] II. Measurement Method 1. Adding samples Rabies virus samples were added to the reaction wells at a rate of 20 viruses per well, 20 μl / well.

[0054] 2. Add cells Add 20 μL of a 5×10⁻⁶ concentration to each well. 5 The microplate was prepared by adding 40 μl of DMEM culture medium (containing 10% serum) to each well, resulting in a total of 80 μl per well. The microplate was then incubated at 37°C and 5% CO2 for 0–24 h. 13 independent parallel plates were set up at 2-hour intervals for dynamic observation.

[0055] 3. Fix After the microplate culture at each time point was completed, the supernatant was discarded, 80 μl of PBS was added to wash once and the plate was dried. 50 μl of 80% acetone pre-cooled to 4℃ was added to each well and the plate was fixed at 4℃ for 30 minutes (or at -30℃ for 10 minutes).

[0056] 4. Antibody incubation Discard the acetone and allow it to evaporate and dry. Add 20 μl of antibody working solution (FITC rabies virus fluorescent antibody) to each well and incubate at 37°C for 30 minutes.

[0057] 5. Observation Discard the liquid, wash the plate three times with PBS, shake dry, add 20 μl of 80% glycerol to each well, and observe under a fluorescence microscope.

[0058] 6. Result Determination An inverted fluorescence microscope was used to continuously observe samples at each time point with the same field of view, with a 2-hour interval between each time point. Under the inverted fluorescence microscope, an overview of the entire well field of view was initially obtained at 50× total magnification (5× objective lens, 10× eyepiece lens) for counting; during imaging, the magnification was switched to 100× total magnification (10× objective lens, 10× eyepiece lens) to ensure that the cell imaging effect met expectations.

[0059] The determination and calculation methods for the virus replication cycle are as follows: (1) Define the incubation time point at which the first typical fluorescent spot (i.e., the fluorescent spot formed in the cell after the virus infects the cell) appears as T1, and the corresponding number of fluorescent spots is A1 (based on the number of cells in which fluorescent spots are observed, reflecting the number of initially infected cells). (2) Subsequent time points are denoted as T. i (i = 2, 3, …, n), corresponding to A fluorophores. i ; (3) When A is satisfied for the first time n When k ≥ k·A1 (k ≥ 2, in this embodiment k = 3), the corresponding time is T. n Then the virus replication cycle T = T n – T1.

[0060] Infected cell ratio = Number of virus-infected cells / Total number of cells (1×10⁻⁶) 4 indivual).

[0061] III. Calculation Results The results of observing the number of fluorescent foci at different incubation time points are shown in Table 2. The results indicate that: The number of fluorescent foci was 0 at the 6-hour time point. The earliest incubation time to show typical fluorescent foci was T1 (T1=8h, the number of fluorescent foci was 18, that is, the number of viruses infecting the cells A1 was about 18). The number of fluorescent foci A8 was 32 at the incubation time T8 (T8=22h). The shortest time point at which fluorescent foci A9 (A9>60) appeared was higher than k·A1 (k=3) was T9 (T9=24h). Therefore, it was determined that the rabies virus had undergone one replication cycle after T1, and the rabies virus replication cycle T was T9-T1=16h.

[0062] Table 2. Observation results of the number of fluorescent foci at different incubation time points.

[0063] Note: All observations at the same time point were conducted under the same field of view and parameters to ensure data comparability; the experiment was repeated 3 times and the results were consistent.

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for determining the viral replication cycle, characterized in that, Cells were infected with the virus to be tested, and cell samples were continuously monitored to observe the number of infected cells. The time point at which infected cells were first observed was recorded as T1, and the corresponding proportion of infected cells was recorded as A1. The monitoring continued until the proportion of infected cells was observed to be greater than or equal to k·A1, and the corresponding time was recorded as T1. n Then the virus replication cycle T = T n T1; k is a real number greater than or equal to 2.

2. The method according to claim 1, characterized in that, The interval between continuous monitoring is 1 to 6 hours.

3. The method according to claim 2, characterized in that, The time interval is 1 to 3 hours.

4. The method according to claim 1, characterized in that, k is any integer among 2, 3, 4, and 5.

5. The method according to claim 1, characterized in that, The multiplicity of infection for the virus is 0.001 to 0.

005.

6. The method according to claim 1, characterized in that, The virus is a virus that can be recognized by a specific fluorescent antibody; preferably, the virus is a virus of the Orthomyxoviridae family, Paramyxoviridae family, Herpesviridae family, Coronaviridae family, Retroviridae family, or Rhabdoviridae family.

7. The method according to claim 1, characterized in that, The cells include at least one of BHK-21 cells, BSR cells, Vero cells, MDCK cells, or HEK293T cells.

8. The method according to claim 1, characterized in that, Cell samples were labeled with virus-specific fluorescent antibodies for quantitative observation.

9. The method according to claim 8, characterized in that, The specific fluorescent antibody is a monoclonal or polyclonal antibody that specifically binds to viral nucleoproteins.

10. The method according to claim 9, characterized in that, The specific fluorescent antibody is any one of FITC, TRITC, Cy3, and Cy5.

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