Separation method of porcine reproductive and respiratory syndrome virus and porcine circovirus

Abstract

The invention discloses a method for separating porcine reproductive and respiratory syndrome virus and porcine circovirus, and belongs to the technical field of virus separation. The separation method comprises the following steps: preparing a virus positive sample into homogenate, and centrifuging to obtain virus liquid; mixing the virus solution with polypropylene glycol and a cholesterol solution to obtain a virus infection solution; inoculating cells with the virus infection solution, and co-culturing in a virus culture solution; and after co-culture is finished, freezing and thawing the cell culture for three times to serve as an F1 generation, subculturing to an F3 generation according to the steps (1)-(3), and harvesting venom. According to the method, polypropylene glycol and cholesterol are added in the virus separation process, the sensitivity of cells to viruses is improved, noradrenaline is added into a virus culture solution, a cell signal channel is adjusted, virus proliferation and release can be promoted, and the purpose of rapidly separating the viruses is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of virus isolation, and particularly to a method for isolating porcine reproductive and respiratory syndrome virus and porcine circovirus. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), characterized by reproductive disorders, premature births, abortions, stillbirths, mummified fetuses in sows and respiratory symptoms in piglets. For vaccine evaluation and development, pathogenic mechanism research of epidemic strains, virus-host cell interaction research and diagnostic method development, all of the above work rely on PRRSV isolated strains. The isolation of PRRSV usually depends on specific cell lines (such as PAM, CL2621, Marc145, etc.), but the adaptability of different strains to cells varies significantly. Although PAM cells are highly sensitive, they are primary cells with disadvantages such as cumbersome preparation, large differences between cell batches, inability to guarantee purity and high cost; Marc145 cells are subculture cells with advantages such as small batch differences, high purity and low cost. However, due to the high genetic diversity of PRRSV field strains, the sensitivity of Marc145 cells to field strains is reduced, making it difficult to isolate strains.

[0003] Porcine circovirus (PCV) belongs to the genus Circovirus of the family Circoviridae, and its gene types include PCV1, PCV2, PCV3 and PCV4. Porcine circovirus type 2 and type 3 are the main pathogens of porcine circovirus disease. PCV infection causes various diseases such as postweaning multisystemic wasting syndrome and porcine dermatitis nephropathy syndrome, causing a huge economic impact on the pig industry. PCV virus can proliferate in porcine-derived cells such as PK15, Vero and ST cells. PK15 cells are the best cells for in vitro isolation and culture of PCV, but the infected cells do not produce lesions, and the replication ability of PCV2 in host cells depends on the expression level of proteins in the cell growth cycle. When PCV is inoculated into PK15 cells, due to the different division cycles of the cells, the virus often cannot fully infect the cells, resulting in a low virus titer.

[0004] Therefore, there is an urgent need for a method for quickly isolating pathogens for porcine reproductive and respiratory syndrome virus and porcine circovirus, which is of great significance for the research of diseases. Summary of the Invention

[0005] The object of the present invention is to provide a method for isolating porcine reproductive and respiratory syndrome virus and porcine circovirus, so as to solve the problems existing in the above-mentioned prior art. By adding polypropylene glycol and cholesterol during the virus isolation process, the present invention enhances the sensitivity of cells to the virus. Norepinephrine is added to the virus culture medium to adjust the cell signaling pathway, which can promote the proliferation and release of the virus in cells, achieving the purpose of rapid virus isolation and providing technical support for the research on epidemic strains of porcine reproductive and respiratory syndrome virus and porcine circovirus.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for isolating a virus, comprising the following steps:

[0008] (1) Prepare a homogenate from the virus-positive sample, and obtain a virus solution by centrifugation.

[0009] (2) Mix the virus solution with polypropylene glycol and a cholesterol solution to obtain a virus infection solution.

[0010] (3) Inoculate the virus infection solution onto cells and co-culture them in a virus culture medium.

[0011] (4) After the co-culture is completed, freeze-thaw the cell culture 3 times to obtain the F1 generation, and subculture according to steps (1)-(3) until the F3 generation, and harvest the virus liquid.

[0012] The composition of the virus culture medium is: basal medium + 1%-3% newborn bovine serum + 150-250 μg / mL cholesterol + 4-6 μmol / L calcium chloride + 2-10 μmol / L norepinephrine.

[0013] Optionally, the virus includes porcine reproductive and respiratory syndrome virus or porcine circovirus.

[0014] Optionally, the centrifugation is at 12000 rpm for 8-12 minutes.

[0015] Further, the mass concentration of polypropylene glycol in the virus infection solution is 5%-10%, and the concentration of cholesterol is 150-250 μg / mL.

[0016] Optionally, the inoculation amount of the virus infection solution is 8%-12%.

[0017] Optionally, the polypropylene glycol is polypropylene glycol 200 or polypropylene glycol 400; the cells are Marc145 cells or PK15 cells.

[0018] Further, the process of co-culturing in the virus culture medium is as follows: after inoculating the virus-infected solution onto the cells, add the virus culture medium, let it stand and adsorb for 30 - 60 minutes, then discard the mixture, add the virus culture medium again, and culture it in an environment of 37°C and 5% CO2 for 72 - 96 hours.

[0019] Optionally, the basal medium is RPMI 1640 medium or DMEM medium.

[0020] The present invention also provides a virus culture medium for isolating and culturing viruses. The composition of the virus culture medium is: basal medium + 1% - 3% newborn bovine serum + 150 - 250 μg / mL cholesterol + 4 - 6 μmol / L calcium chloride + 2 - 10 μmol / L norepinephrine;

[0021] The basal medium is RPMI 1640 medium or DMEM medium.

[0022] The present invention also provides the application of the above virus culture medium in clinical virus isolation and culture or increasing virus titer. The viruses include porcine reproductive and respiratory syndrome virus or porcine circovirus.

[0023] The present invention discloses the following technical effects:

[0024] By adding polypropylene glycol and cholesterol during the virus isolation process, the present invention enhances the sensitivity of cells to the virus. Norepinephrine is added to the virus culture medium to adjust the cell signaling pathway, promote the proliferation and release of viruses in cells, and significantly improve the virus proliferation efficiency, solving the problems of low isolation rate and long cycle in conventional isolation methods. The present invention establishes a rapid and efficient virus isolation method, which can significantly improve the virus isolation efficiency. The isolation cycle is shortened from 6 - 8 weeks of the conventional method to 2 - 3 weeks, and it has the advantages of high isolation rate, short cycle, and strong specificity, achieving the purpose of rapid virus isolation, and providing technical support for the research on epidemic strains of porcine reproductive and respiratory syndrome virus and porcine circovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 For the F1 generation of porcine reproductive and respiratory syndrome virus cultured in Marc145 cells for 96 hours;

[0027] Figure 2For the F2 generation of porcine reproductive and respiratory syndrome virus cultured in Marc145 cells for 96 hours;

[0028] Figure 3 For the F3 generation of porcine reproductive and respiratory syndrome virus cultured in Marc145 cells for 72 hours. Detailed implementation manners

[0029] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0033] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0034] The technical concept of the present invention is as follows:

[0035] In the present invention, by adding polypropylene glycol and cholesterol during the virus isolation process, the sensitivity of cells to the virus is enhanced. Norepinephrine is added to the virus culture solution to adjust the cell signaling pathway, promoting the proliferation and release of the virus within the cells. The present invention has established a rapid and efficient virus isolation method, which has the advantages of high isolation rate, short cycle, and strong specificity, achieving the purpose of rapidly isolating the virus, and providing technical support for the research on epidemic strains of porcine reproductive and respiratory syndrome virus and porcine circovirus.

[0036] Example 1

[0037] This example provides a method for isolating porcine reproductive and respiratory syndrome virus, and the specific process is as follows:

[0038] 1. The porcine reproductive and respiratory syndrome virus-positive lung or lymph node tissue samples identified are minced with surgical scissors. The tissue samples are mixed with pre-cooled PBS solution at a ratio of 1:5 (w / v), homogenized for 90 seconds, and the processed tissue samples are centrifuged at 12000 rpm for 10 minutes. The supernatant is taken and filtered through a 0.22 μm filter to remove bacteria, and the virus solution preparation is completed.

[0039] 2. After the virus solution is mixed evenly with polypropylene glycol 200 and cholesterol solution, the concentration of polypropylene glycol 200 is 5% wt, and the concentration of cholesterol is 200 μg / mL to obtain a virus infection solution.

[0040] 3. Take Marc145 cells cultured for 72 hours, discard the original culture solution, add the virus infection solution to the cell culture flask at a ratio of 8% (v / v), and simultaneously supplement the virus culture solution (components: DMEM medium, 2% newborn bovine serum, 200 μg / mL cholesterol, 5 μmol / L calcium chloride, 10 μmol / L norepinephrine) with the same volume as the discarded culture solution. After static adsorption for 30 minutes, the mixture is discarded, and the same volume of virus culture solution is supplemented, and it is placed in an incubator at 37°C and 5% CO2 for continued culture.

[0041] 4. After continued culture for 72 hours, the cell culture is frozen and thawed 3 times as the F1 generation. The F1 generation virus solution is collected, and subculture is carried out according to steps 2 - 3 until the F3 generation, and the F2 generation and F3 generation virus solutions are harvested respectively.

[0042] 5. Virus content determination

[0043] The harvested F1 - F3 generation virus solutions are serially diluted 10-fold continuously with DMEM basal culture solution, transferred to a 96-well Marc145 cell culture plate filled with a monolayer at 0.2 mL / well, and cultured in an incubator at 37°C and 5% CO2 for 5 days. The cytopathic effect is observed and recorded every day, and the TCID is calculated by the Reed-Muench method 50 ;

[0044] 6. RT-qPCR Detection

[0045] A commercial porcine reproductive and respiratory syndrome virus detection kit (purchased from Nanjing Novozymes Biotech Co., Ltd.) was used for nucleic acid detection. Viral RNA was extracted using an RNA extraction kit, and a 25 μL reaction system was adopted: 20 μL of PCR reaction solution and 5 μL of RNA. The reaction procedure was: 15 min at 55 °C, 30 s at 95 °C; 10 s at 95 °C, 30 s at 60 °C, for a total of 40 cycles. At the same time, negative and positive controls were set, and fluorescence was collected from the VIC channel.

[0046] 7. Test Results

[0047] Table 1 Results of virus content determination

[0048] Virus passage number <![CDATA[Virus titer (Lg10TCID 50 / mL)]]> F1 Less than 3.0 F2 4.0 F3 6.5

[0049] Table 2 RT-qPCR detection results

[0050]

[0051]

[0052] As can be seen from the results in Table 1 and Table 2, as the virus passage number increases, the virus titer gradually increases. After only 3 passages of culture, the titer of porcine reproductive and respiratory syndrome virus can reach 10 6.5 TCID 50 / mL. The state diagrams of the F1 generation virus cultured in Marc145 cells for 96 hours are as shown in Figure 1 ; the state diagrams of the F2 generation virus cultured in Marc145 cells for 96 hours are as shown in Figure 2 ; the state diagrams of the F3 generation virus cultured in Marc145 cells for 72 hours are as shown in Figure 3 .

[0053] Comparative Example 1

[0054] In this comparative example, the factors affecting virus isolation efficiency were verified by setting up control experiments. The following control groups were set:

[0055] Control Group 1: On the basis of Example 1, polypropylene glycol 200 and cholesterol in step 2 were omitted, and the remaining steps were the same as those in Example 1.

[0056] Control Group 2: On the basis of Example 1, norepinephrine in the virus culture medium was omitted, and the remaining steps were the same as those in Example 1.

[0057] The F1-F3 virus solutions harvested from Control Groups 1-2 were subjected to virus content determination and RT-qPCR detection, and the test results are as follows:

[0058] Table 3 Results of virus content determination

[0059]

[0060] Table 4 RT-qPCR detection results

[0061]

[0062] Example 2 Isolation of Porcine circovirus

[0063] 1. Cut the lung or lymph node tissue samples identified as positive for Porcine circovirus into small pieces with surgical scissors. Mix the tissue samples with pre-cooled PBS solution at a ratio of 1:5 (w / v), perform homogenization for 90 seconds, centrifuge the treated tissue samples at 12000 rpm for 10 minutes, and take the supernatant. After filtering and sterilizing through a 0.22 μm filter, the virus solution preparation is completed.

[0064] 2. After mixing the virus solution, polypropylene glycol 400, and cholesterol solution evenly, make the concentration of polypropylene glycol 400 10% wt and the concentration of cholesterol 200 μg / mL to obtain the virus infection solution.

[0065] 3. Take Marc145 cells cultured for 72 hours, discard the original culture medium, add the virus infection solution to the cell culture flask at a ratio of 12% (v / v), and simultaneously supplement the virus culture medium (components: RPMI 1640 medium, 2% newborn bovine serum, 200 μg / mL cholesterol, 5 μmol / L calcium chloride, 2 μmol / L norepinephrine) with the same volume as the discarded culture medium. After adsorption for 60 minutes, discard the mixture, supplement with an equal volume of virus culture medium, and place in an incubator at 37°C and 5% CO2 for continued culture.

[0066] 4. After continued culture for 72 hours, freeze-thaw the cell culture three times as the F1 generation, collect the F1 generation virus solution, and subculture according to steps 2-3 until the F3 generation. Harvest the F2 generation and F3 generation virus solutions respectively.

[0067] 5. Virus content determination

[0068] Dilute the harvested F1-F3 generation virus solutions continuously 10-fold with DMEM basal culture medium, transfer 0.2 mL / well into a 96-well PK15 cell culture plate covered with a monolayer, place in an incubator at 37°C and 5%, and after culturing for 48 h, detect by the IFA method and calculate the TCID according to the Reed-Muench method 50 .

[0069] 6. RT-qPCR detection

[0070] Nucleic acid detection was carried out using a commercial porcine circovirus detection kit (purchased from Hunan Guanmu Biotechnology Co., Ltd.). Viral DNA was extracted using a DNA extraction kit, and a 25 μL reaction system was used: 25 μL of PCR reaction solution, 15 μL of Oligo Mix, and 10 μL of DNA. The reaction program was: 50 °C for 2 min, 95 °C for 120 s; 95 °C for 15 s, 60 °C for 30 s, for a total of 45 cycles. At the same time, negative and positive controls were set, and fluorescence in the FAM channel was collected.

[0071] 7. Test Results

[0072] Table 5 Results of virus titer determination

[0073] Virus passage number <![CDATA[Virus titer (Lg10TCID 50 / mL)]]> F1 Less than 3.0 F2 3.5 F3 5.0

[0074] Table 6 RT-qPCR detection results

[0075] Virus passage number Nucleic acid test (Ct) F1 25.65 F2 16.58 F3 12.56

[0076] As can be seen from Table 5 and Table 6, as the virus passage number increases, the virus titer gradually increases. After only 3 passages of culture, the titer of porcine circovirus can reach 10 5 TCID 50 / mL.

[0077] Comparative Example 2

[0078] In this comparative example, the factors affecting virus isolation were verified by setting up control experiments. The control groups were set as follows:

[0079] Control Group 3: On the basis of Example 2, polypropylene glycol 400 and cholesterol in Step 2 were omitted, and the remaining steps were the same as in Example 2.

[0080] Control Group 4: On the basis of Example 2, norepinephrine in the virus culture medium was omitted, and the remaining steps were the same as in Example 2.

[0081] The F1-F3 virus fluids harvested from Control Groups 3-4 were subjected to virus titer determination and RT-qPCR detection. The test results are as follows:

[0082] Table 7 Results of virus titer determination

[0083]

[0084] Table 8 RT-qPCR detection results

[0085]

[0086] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for isolating a virus, characterized in that, It includes the following steps: (1) Prepare a homogenate from the virus-positive sample, and obtain a virus solution by centrifugation; (2) Mix the virus solution with polypropylene glycol and a cholesterol solution to obtain a virus infection solution; (3) Inoculate the virus infection solution onto cells and co-culture them in a virus culture medium; (4) After the co-culture is completed, freeze-thaw the cell culture 3 times and use it as the F1 generation. Subculture it to the F3 generation according to steps (1)-(3) and harvest the venom; The composition of the virus culture medium is: basal medium + 1%-3% newborn bovine serum + 150-250 μg / mL cholesterol + 4-6 μmol / L calcium chloride + 2-10 μmol / L norepinephrine.

2. The separation method according to claim 1, characterized in that, The virus includes porcine reproductive and respiratory syndrome virus or porcine circovirus.

3. The separation method according to claim 1, wherein, The centrifugation is at 12000 rpm for 8-12 minutes.

4. The separation method according to claim 1, wherein The mass concentration of polypropylene glycol in the virus infection solution is 5%-10%, and the concentration of cholesterol is 150-250 μg / mL.

5. The separation method according to claim 1, characterized in that, The inoculation amount of the virus infection solution is 8%-12%.

6. The separation method according to claim 1, characterized in that The polypropylene glycol is polypropylene glycol 200 or polypropylene glycol 400; the cells are Marc145 cells or PK15 cells.

7. The separation method according to claim 1, characterized in that The process of co-culturing in the virus culture medium is as follows: after inoculating the virus infection solution onto the cells, add the virus culture medium, let it stand and adsorb for 30-60 minutes, then discard the mixture, replenish the virus culture medium, and place it in an environment of 37°C and 5% CO2 for 72-96 hours.

8. The separation method according to claim 1, wherein The basal medium is RPMI 1640 medium or DMEM medium.

9. A virus culture medium for separating and culturing viruses, characterized in that, The composition of the virus culture medium is: basal medium + 1%-3% newborn bovine serum + 150-250 μg / mL cholesterol + 4-6 μmol / L calcium chloride + 2-10 μmol / L norepinephrine; The basal medium is RPMI 1640 medium or DMEM medium.

10. Use of the virus culture solution according to claim 9 in clinical virus isolation and culture or in increasing virus titer, characterized in that, The virus includes porcine reproductive and respiratory syndrome virus or porcine circovirus.

Images