Method for efficient isolation of human parainfluenza virus based on suspension MDCK cells

Abstract

This invention discloses a method for efficiently isolating human parainfluenza virus based on suspended MDCK cells. By using the fully suspended passaged MDCK cell line for human parainfluenza virus isolation and culture and optimizing the inoculation process, single-generation virus amplification can be completed within 3 days, significantly shortening the cycle, improving virus isolation efficiency and success rate compared to traditional adherent culture methods, and enhancing experimental reproducibility. This method not only demonstrates good isolation and culture capabilities for the major subtypes of human parainfluenza virus, but also shows good adaptability to clinical samples from different sources and with different initial loads. In addition, this culture system has linear scale-up potential, is not limited by the surface area of ​​adherent culture, and can meet the large-scale virus seed preparation needs from clinical testing to vaccine mass production, providing key technical support for the integrated process from human parainfluenza virus isolation to vaccine production.

Description

Technical Field

[0001] This invention belongs to the field of human parainfluenza virus isolation technology, and more specifically, relates to a method for efficiently isolating human parainfluenza virus based on suspended MDCK cells. Background Technology

[0002] Human parainfluenza virus (HPIV) is a major pathogen causing acute respiratory infections in infants, the elderly, and immunocompromised individuals, and its clinical disease burden is second only to respiratory syncytial virus (RSV) globally. Of the four known serotypes, HPIV-3 infection is the most prevalent and pathogenic, and is one of the leading causes of severe bronchiolitis and pneumonia in children. Currently, there are no specific antiviral drugs or approved vaccines for HPIV infection. Therefore, establishing rapid and reliable virus isolation and culture techniques is crucial for disease diagnosis, epidemiological surveillance, and vaccine development.

[0003] Virus isolation and culture, considered the "gold standard" for pathogen diagnosis, provides intact, infective viral particles and is an indispensable foundation for analyzing viral characteristics, screening vaccine candidates, and evaluating antiviral drugs. Currently, conventional HPIV isolation techniques primarily rely on adherent cell culture systems (such as LLC-MK2 and HEp-2 cells) and centrifugation-enhanced culture methods (such as the shell vial assay). However, these methods have significant limitations: adherent cell culture is cumbersome, requiring repeated trypsin digestion, and the culture cycle for a single generation of virus typically lasts 5 to 8 days, with limited throughput and difficulty in standardization; while centrifugation can shorten detection time, its sensitivity is unstable, and the viral load obtained is usually insufficient to support subsequent amplification and research needs. More importantly, existing cell systems suitable for virus culture also have many shortcomings. For example, primary cells (such as monkey kidney cells) are difficult to obtain, costly, and exhibit significant batch-to-batch variability; while some continuously passaged cell lines may pose potential tumorigenic risks and are unsuitable for the production of vaccines and other biological products. Even when using existing cells for culture, the resulting virus titers are often too low to meet the needs of high-quality vaccine preparation or in-depth research. Summary of the Invention

[0004] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a method for efficiently isolating human parainfluenza virus based on suspended MDCK cells. By employing the fully suspended passaged MDCK cell line for human parainfluenza virus isolation and culture, and optimizing the inoculation process, single-generation virus amplification can be completed within 3 days. This significantly shortens the cycle, improves virus isolation efficiency and success rate compared to traditional adherent culture methods, and enhances experimental reproducibility. This method not only demonstrates excellent isolation and culture capabilities for all major subtypes of human parainfluenza virus but also exhibits good adaptability to clinical samples from different sources and with different initial viral loads. Furthermore, this culture system possesses linear scale-up potential, is not limited by adherent culture surface area, and can meet the large-scale virus seed preparation requirements from clinical testing to vaccine mass production, providing key technical support for the integrated process from human parainfluenza virus isolation to vaccine production.

[0005] To achieve the above objectives, the present invention employs the following technical solution: A method for efficiently isolating human parainfluenza virus based on suspended MDCK cells includes the following steps: Step 1, Preparation of suspension MDCK cells: Revive the frozen suspension MDCK cells, culture them in an incubator with shaking using cell growth medium, count the viable cells and passage them. Step 2, virus inoculation tube preparation: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, adjust the cell concentration and dispense into centrifuge tubes; add TPCK trypsin and triple antibody solution to each tube, mix well and set aside. Step 3, Virus inoculation and culture: After taking the clinical sample out of -80 ℃ and freezing and thawing it once, mix it thoroughly. Add the treated sample to the prepared virus inoculation tube and mix well. Place the inoculated centrifuge tube in an incubator and shake to culture. Step 4, Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge and collect the supernatant, which is the first-generation virus solution. Repeat the above operation step 3 for passage again to obtain cultured human parainfluenza virus.

[0006] Preferably, in step 1, the cell culture temperature is 37 ℃, the CO2 concentration in the incubator is 2~10%, and the shaking speed is 120~180 rpm.

[0007] Preferably, in step 1, live cell counting and passage should be performed every 2-3 days, maintaining a cell concentration of 0.8~1.2×10⁻⁶. 6 / mL, ensuring a viable cell rate >95%.

[0008] Preferably, in step 2, the cell concentration is adjusted to 3 × 10⁻⁶. 6 / mL~5×10 6 / mL.

[0009] Preferably, in step 2, each inoculation tube contains 10 mL to 30 mL of cell suspension, 100 μL to 300 μL of 1% tri-antibody solution, and 5 to 40 mg / L of TPCK trypsin.

[0010] Preferably, in step 3, the amount of clinical sample added is 0.5 mL to 1 mL.

[0011] Preferably, the culture temperature is 33 ℃~35 ℃, the CO2 concentration in the incubator is 2~10%, the shaking speed is 180~220 rpm, and the culture time is 3~5 days.

[0012] Preferably, in step 4, the centrifugation speed is 2000~3000 rpm and the time is 5~10 min.

[0013] Preferably, in step 4, the virus solution from the first passage is used as the seed virus for the next generation of virus culture, and the cells are passaged 2 to 5 times.

[0014] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: 1. This invention utilizes the fully suspended passaged cell line MDCK for the isolation and culture of human parainfluenza virus, combined with an optimized inoculation process. This allows for efficient amplification of a single-generation virus within 3 days, resulting in high virus titers in the harvested broth. Compared to traditional methods (which typically require 5-8 days per generation), this significantly shortens the isolation cycle and improves isolation efficiency. It avoids the drawbacks of traditional adherent cell methods, such as frequent trypsin digestion, cumbersome operations, and susceptibility to contamination. This effectively improves the reproducibility and stability of the experiment. The system process is stable and has good reproducibility, laying a reliable technical foundation for large-scale, homogeneous virus culture.

[0015] 2. The method of this invention not only demonstrates excellent isolation and culture capabilities for the major subtypes of human parainfluenza virus (types 1, 2, and 3), but also exhibits good adaptability to clinical samples from different sources and with different initial viral loads. This proves that the method is not optimized for individual strains, but rather a robust platform technology with broad applicability.

[0016] 3. The culture system of this invention has good linear scale-up potential, fundamentally overcoming the bottleneck of adherent culture in scale-up production. It is easy to monitor, control and standardize the process. This method can not only meet the testing needs of clinical laboratories, but also has the potential to connect to the large-scale virus seed bank preparation stage required in vaccine research and development and production, providing key technical support for realizing the whole process integration from virus isolation to vaccine production. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the basic process for isolating human parainfluenza virus according to an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the isolation rate of human parainfluenza virus from clinical samples in Examples 7-9 of the present invention; Figure 3 This is a schematic diagram of the PCR results of the cultures isolated and cultured for one generation from clinical samples of Example 10 and Comparative Example 1 of the present invention. Figure 4 This is a schematic diagram of the PCR results of cultures from different subtypes of HPIV clinical samples isolated and cultured for one generation, as shown in Example 10 and Comparative Example 1 of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0019] The detection and identification methods for human parainfluenza virus obtained in the examples and comparative examples are as follows: (1) Rt-PCR: The CT values ​​of human parainfluenza virus before and after isolation were detected using a quadruple nucleic acid detection kit for human parainfluenza virus (types 1, 2, 3, and 4) provided by Shanghai Berger Medical Technology Co., Ltd.

[0020] (2) HA titration: 50 μL of sample was diluted sequentially by 2, 4, 8, 16, 32, 64, and 128 times, and then an equal volume of 50 μL of 0.75% guinea pig erythrocytes was added. After incubation at 4 ℃ for 1 h, agglutination was observed. Agglutination was indicated by the formation of chain-like, petal-like, or small clump-like clusters of erythrocytes on the surface of a monolayer that did not move with shaking of the cell plate. The maximum dilution factor that caused agglutination was recorded.

[0021] (3) The criteria for virus identification are: a CT value less than 13 and an HA dilution factor greater than or equal to 4 are considered as successful virus isolation.

[0022] Example 1 The materials used in Example 1 of this invention are sourced from the following sources: 1. Cells: Suspension MDCK cells, provided by Shanghai Berger Medical Technology Co., Ltd.

[0023] 2. Virus: Human parainfluenza virus type 1 (HPIV-1), sampled from a pharyngeal swab from a febrile patient, provided by Wuhan Children's Hospital.

[0024] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0025] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0026] 5. Triple Antibiotic Solution: Contains the antibiotics penicillin, streptomycin, and amphotericin B, provided by Shanghai Berger Medical Technology Co., Ltd.

[0027] The method for culturing human parainfluenza virus type 1 according to an embodiment of the present invention is as follows: (1) Preparation of suspension MDCK cells: Using conical shake flasks containing cell growth medium, culture at 37 ℃ and 5% CO2 with shaking at 120 rpm; count viable cells and passage every 2-3 days, maintaining a cell concentration of 0.8-1.2 × 10⁻⁶ cells / day. 6 Cells / mL, ensuring a viable cell rate >95%.

[0028] (2) Preparation of virus inoculation tubes: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, and adjust the cell concentration to 3×10⁻⁶. 6 The cells were aliquoted into centrifuge tubes, with 10 mL of cell suspension and 100 μL of 1% triple antibody solution added to each tube. TPCK trypsin at final concentrations of 5 mg / L, 20 mg / L, and 40 mg / L were added to the inoculation centrifuge tubes and mixed thoroughly for later use.

[0029] (3) Virus inoculation and culture: Take out the clinical sample, freeze and thaw it once, and mix it thoroughly on a shaker; add 0.5 mL of the treated virus sample to each of the prepared virus inoculation tubes and mix gently. Place the inoculated centrifuge tubes in an incubator at 34℃ and 5% CO2 and culture at 180 rpm for 3 days.

[0030] (4) Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge at 3000 rpm for 10 min and collect the supernatant, which is the first generation of virus solution. Repeat the above operation for the second generation.

[0031] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0032] Example 2 The materials used in Example 2 of this invention are sourced from the following sources: 1. Cells: Suspension MDCK cells, provided by Shanghai Berger Medical Technology Co., Ltd.

[0033] 2. Virus: Human parainfluenza virus type 1, sampled from a pharyngeal swab from a febrile patient, provided by Wuhan Children's Hospital.

[0034] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0035] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0036] 5. Triple Antibiotic Solution: Contains the antibiotics penicillin, streptomycin, and amphotericin B, provided by Shanghai Berger Medical Technology Co., Ltd.

[0037] The method for culturing human parainfluenza virus type 1 according to an embodiment of the present invention is as follows: (1) Preparation of suspension MDCK cells: Using conical shake flasks containing cell growth medium, culture at 37 ℃ and 5% CO2 with shaking at 120 rpm; count viable cells and passage every 2-3 days, maintaining a cell concentration of 0.8-1.2 × 10⁻⁶ cells / day. 6 Cells / mL, ensuring a viable cell rate >95%.

[0038] (2) Preparation of virus inoculation tubes: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, and adjust the cell concentration to 3×10⁻⁶. 6 The cells were aliquoted into centrifuge tubes, with 10 mL of cell suspension and 100 μL of 1% triple antibody solution added to each tube. TPCK trypsin at final concentrations of 5 mg / L, 20 mg / L, and 40 mg / L were added to the inoculation centrifuge tubes and mixed thoroughly for later use.

[0039] (3) Virus inoculation and culture: Take out the clinical sample, freeze and thaw it once, and mix it thoroughly on a shaker; add 0.5 mL of the treated sample to each of the prepared virus inoculation tubes and mix gently. Place the inoculated centrifuge tubes in an incubator at 34 ℃ and 5% CO2 and culture at 180 rpm for 3 days.

[0040] (4) Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge at 3000 rpm for 10 min and collect the supernatant, which is the first generation of virus solution. Repeat the above operation for the second generation.

[0041] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0042] Example 3 The materials used in Example 3 of this invention are sourced from the following sources: 1. Cells: Suspension MDCK cells, provided by Shanghai Berger Medical Technology Co., Ltd.

[0043] 2. Virus: Human parainfluenza virus type 2 (HPIV-2), sampled from a pharyngeal swab from a febrile case, provided by Wuhan Children's Hospital.

[0044] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0045] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0046] 5. Triple Antibiotic Solution: Contains the antibiotics penicillin, streptomycin, and amphotericin B, provided by Shanghai Berger Medical Technology Co., Ltd.

[0047] The method for culturing human parainfluenza virus type 2 according to an embodiment of the present invention is as follows: (1) Preparation of suspension MDCK cells: Using conical shake flasks containing cell growth medium, culture at 37 ℃ and 5% CO2 with shaking at 120 rpm; count viable cells and passage every 2-3 days, maintaining a cell concentration of 0.8-1.2 × 10⁻⁶ cells / day. 6 Cells / mL, ensuring a viable cell rate >95%.

[0048] (2) Preparation of virus inoculation tubes: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, and adjust the cell concentration to 3×10⁻⁶. 6 The cells were aliquoted into centrifuge tubes, with 10 mL of cell suspension and 100 μL of 1% triple antibody solution added to each tube. TPCK trypsin at final concentrations of 5 mg / L, 20 mg / L, and 40 mg / L were added to the inoculation centrifuge tubes and mixed thoroughly for later use.

[0049] (3) Virus inoculation and culture: Take out the clinical sample, freeze and thaw it once, and mix it thoroughly on a shaker; add 0.5 mL of the treated virus sample to each of the prepared virus inoculation tubes and mix gently. Place the inoculated centrifuge tubes in an incubator at 34℃ and 5% CO2 and culture at 180 rpm for 3 days.

[0050] (4) Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge at 3000 rpm for 10 min and collect the supernatant, which is the first generation of virus solution. Repeat the above operation for the second generation.

[0051] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0052] Example 4 The materials used in Example 4 of this invention are sourced from the following sources: 1. Cells: Suspension MDCK cells, provided by Shanghai Berger Medical Technology Co., Ltd.

[0053] 2. Virus: Human parainfluenza virus type 2, sampled from a pharyngeal swab from a febrile patient, provided by Wuhan Children's Hospital.

[0054] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0055] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0056] 5. Triple Antibiotic Solution: Contains the antibiotics penicillin, streptomycin, and amphotericin B, provided by Shanghai Berger Medical Technology Co., Ltd.

[0057] The method for culturing human parainfluenza virus type 2 according to an embodiment of the present invention is as follows: (1) Preparation of suspension MDCK cells: Using conical shake flasks containing cell growth medium, culture at 37 ℃ and 5% CO2 with shaking at 120 rpm; count viable cells and passage every 2-3 days, maintaining a cell concentration of 0.8-1.2 × 10⁻⁶ cells / day. 6 Cells / mL, ensuring a viable cell rate >95%.

[0058] (2) Preparation of virus inoculation tubes: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, and adjust the cell concentration to 3×10⁻⁶. 6 The cells were aliquoted into centrifuge tubes, with 10 mL of cell suspension and 100 μL of 1% triple antibody solution added to each tube. TPCK trypsin at final concentrations of 5 mg / L, 20 mg / L, and 40 mg / L were added to the inoculation centrifuge tubes and mixed thoroughly for later use.

[0059] (3) Virus inoculation and culture: Take out the clinical sample, freeze and thaw it once, and mix it thoroughly on a shaker; add 0.5 mL of the treated sample to each of the prepared virus inoculation tubes and mix gently. Place the inoculated centrifuge tubes in an incubator at 34 ℃ and 5% CO2 and culture at 180 rpm for 3 days.

[0060] (4) Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge at 3000 rpm for 10 min and collect the supernatant, which is the first generation of virus solution. Repeat the above operation for the second generation.

[0061] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0062] Example 5 The materials used in Example 5 of this invention are sourced from the following sources: 1. Cells: Suspension MDCK cells, provided by Shanghai Berger Medical Technology Co., Ltd.

[0063] 2. Virus: Human parainfluenza virus type 3 (HPIV-3), sampled from a pharyngeal swab from a febrile patient, provided by Wuhan Children's Hospital.

[0064] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0065] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0066] 5. Triple Antibiotic Solution: Contains the antibiotics penicillin, streptomycin, and amphotericin B, provided by Shanghai Berger Medical Technology Co., Ltd.

[0067] The method for culturing human parainfluenza virus type 3 according to an embodiment of the present invention is as follows: (1) Preparation of suspension MDCK cells: Using conical shake flasks containing cell growth medium, culture at 37 ℃ and 5% CO2 with shaking at 120 rpm; count viable cells and passage every 2-3 days, maintaining a cell concentration of 0.8-1.2 × 10⁻⁶ cells / day. 6 Cells / mL, ensuring a viable cell rate >95%.

[0068] (2) Preparation of virus inoculation tubes: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, and adjust the cell concentration to 3×10⁻⁶. 6 The cells were aliquoted into centrifuge tubes, with 10 mL of cell suspension and 100 μL of 1% triple antibody solution added to each tube. TPCK trypsin at final concentrations of 5 mg / L, 20 mg / L, and 40 mg / L were added to the inoculation centrifuge tubes and mixed thoroughly for later use.

[0069] (3) Virus inoculation and culture: Take out the clinical sample, freeze and thaw it once, and mix it thoroughly on a shaker; add 0.5 mL of the treated sample to each of the prepared virus inoculation tubes and mix gently. Place the inoculated centrifuge tubes in an incubator at 34 ℃ and 5% CO2 and culture at 180 rpm for 3 days.

[0070] (4) Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge at 3000 rpm for 10 min and collect the supernatant, which is the first generation of virus solution. Repeat the above operation for the second generation.

[0071] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0072] Example 6 The materials used in Example 6 of this invention are sourced from the following sources: 1. Cells: Suspension MDCK cells, provided by Shanghai Berger Medical Technology Co., Ltd.

[0073] 2. Virus: Human parainfluenza virus type 3, sampled from a pharyngeal swab from a febrile patient, provided by Wuhan Children's Hospital.

[0074] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0075] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0076] 5. Triple Antibiotic Solution: Contains the antibiotics penicillin, streptomycin, and amphotericin B, provided by Shanghai Berger Medical Technology Co., Ltd.

[0077] The method for culturing human parainfluenza virus type 3 according to an embodiment of the present invention is as follows: (1) Preparation of suspension MDCK cells: Using conical shake flasks containing cell growth medium, culture at 37 ℃ and 5% CO2 with shaking at 120 rpm; count viable cells and passage every 2-3 days, maintaining a cell concentration of 0.8-1.2 × 10⁻⁶ cells / day. 6 Cells / mL, ensuring a viable cell rate >95%.

[0078] (2) Preparation of virus inoculation tubes: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, and adjust the cell concentration to 3×10⁻⁶. 6 The cells were aliquoted into centrifuge tubes, with 10 mL of cell suspension and 100 μL of 1% triple antibody solution added to each tube. TPCK trypsin at final concentrations of 5 mg / L, 20 mg / L, and 40 mg / L were added to the inoculation centrifuge tubes and mixed thoroughly for later use.

[0079] (3) Virus inoculation and culture: Take out the clinical sample, freeze and thaw it once, and mix it thoroughly on a shaker; add 0.5 mL of the treated sample to each of the prepared virus inoculation tubes and mix gently. Place the inoculated centrifuge tubes in an incubator at 34 ℃ and 5% CO2 and culture at 180 rpm for 3 days.

[0080] (4) Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge at 3000 rpm for 10 min and collect the supernatant, which is the first generation of virus solution. Repeat the above operation for the second generation.

[0081] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0082] Table 1. Effects of changes in pancreatic enzyme concentration on the isolation of second-generation human parainfluenza virus (HPIV) types 1-3. Table 1 shows that the success rate of human parainfluenza virus (HIV) isolation was significantly correlated with the concentration of TPCK trypsin. Low concentrations (5 mg / L) were insufficient to support effective viral replication; high concentrations (40 mg / L) may inhibit cell or viral activity; while moderate concentrations (20 mg / L) achieved optimal viral amplification and hemagglutination activity in most samples. This indicates that, under the current system, a TPCK trypsin concentration of 20 mg / L is most favorable for HPIV infection and replication in the culture system. Therefore, subsequent HIV culture passages were performed at this concentration for virus isolation and culture.

[0083] Example 7 The materials used in Example 7 of this invention are sourced from the following sources: 1. Cells: Suspension MDCK cells, provided by Shanghai Berger Medical Technology Co., Ltd.

[0084] 2. Virus: Human parainfluenza virus type 1, sampled from a pharyngeal swab from a febrile patient, provided by Wuhan Children's Hospital.

[0085] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0086] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0087] 5. Triple Antibiotic Solution: Contains the antibiotics penicillin, streptomycin, and amphotericin B, provided by Shanghai Berger Medical Technology Co., Ltd.

[0088] In this embodiment of the invention, viral isolation and culture were performed on pharyngeal swab samples from 9 patients diagnosed as HPIV-1 positive. The specific culture method is as follows: (1) Preparation of suspension MDCK cells: Using conical shake flasks containing cell growth medium, culture at 37 ℃ and 5% CO2 with shaking at 120 rpm; count viable cells and passage every 2-3 days, maintaining a cell concentration of 0.8-1.2 × 10⁻⁶ cells / day. 6 Cells / mL, ensuring a viable cell rate >95%.

[0089] (2) Preparation of virus inoculation tubes: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, and adjust the cell concentration to 3×10⁻⁶.6 The cells were aliquoted into centrifuge tubes, with 10 mL of cell suspension and 100 μL of 1% triple antibody solution added to each tube. TPCK trypsin at a final concentration of 20 mg / L was added to the inoculation centrifuge tubes and mixed thoroughly for later use.

[0090] (3) Virus inoculation and culture: Take out the clinical sample, freeze and thaw it once, and mix it thoroughly on a shaker; add 0.5 mL of the treated sample to each of the prepared virus inoculation tubes and mix gently. Place the inoculated centrifuge tubes in an incubator at 34 ℃ and 5% CO2 and culture at 180 rpm for 3 days.

[0091] (4) Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge at 3000 rpm for 10 min and collect the supernatant, which is the first generation of virus solution. Repeat the above operation for multiple generations.

[0092] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0093] Table 2. Isolation efficiency of the method of the present invention for HPIV-1 virus. Example 8 The materials used in Example 8 of this invention are sourced from the following sources: 1. Cells: Suspension MDCK cells, provided by Shanghai Berger Medical Technology Co., Ltd.

[0094] 2. Virus: Human parainfluenza virus type 2, sampled from a pharyngeal swab from a febrile patient, provided by Wuhan Children's Hospital.

[0095] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0096] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0097] 5. Triple Antibiotic Solution: Contains the antibiotics penicillin, streptomycin, and amphotericin B, provided by Shanghai Berger Medical Technology Co., Ltd.

[0098] In this embodiment of the invention, viral isolation and culture were performed on pharyngeal swab samples from 5 patients diagnosed as HPIV-2 positive. The specific culture method is as follows: (1) Preparation of suspension MDCK cells: Using conical shake flasks containing cell growth medium, culture at 37 ℃ and 5% CO2 with shaking at 120 rpm; count viable cells and passage every 2-3 days, maintaining a cell concentration of 0.8-1.2 × 10⁻⁶ cells / day. 6Cells / mL, ensuring a viable cell rate >95%.

[0099] (2) Preparation of virus inoculation tubes: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, and adjust the cell concentration to 3×10⁻⁶. 6 The cells were aliquoted into centrifuge tubes, with 10 mL of cell suspension and 100 μL of 1% triple antibody solution added to each tube. TPCK trypsin at a final concentration of 20 mg / L was added to the inoculation centrifuge tubes and mixed thoroughly for later use.

[0100] (3) Virus inoculation and culture: Take out the clinical sample, freeze and thaw it once, and mix it thoroughly on a shaker; add 0.5 mL of the treated sample to each of the prepared virus inoculation tubes and mix gently. Place the inoculated centrifuge tubes in an incubator at 34 ℃ and 5% CO2 and culture at 180 rpm for 3 days.

[0101] (4) Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge at 3000 rpm for 10 min and collect the supernatant, which is the first generation of virus solution. Repeat the above operation for multiple generations.

[0102] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0103] Table 3. Isolation efficiency of the method of the present invention for HPIV-2 virus. Example 9 The materials used in Example 9 of this invention are sourced from the following sources: 1. Cells: Suspension MDCK cells, provided by Shanghai Berger Medical Technology Co., Ltd.

[0104] 2. Virus: Human parainfluenza virus type 3, sampled from a pharyngeal swab from a febrile patient, provided by Wuhan No. 1 Hospital.

[0105] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0106] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0107] 5. Triple Antibiotic Solution: Contains the antibiotics penicillin, streptomycin, and amphotericin B, provided by Shanghai Berger Medical Technology Co., Ltd.

[0108] In this embodiment of the invention, viral isolation and culture were performed on pharyngeal swab samples from 9 patients diagnosed as HPIV-3 positive. The specific culture method is as follows: (1) Preparation of suspension MDCK cells: Using conical shake flasks containing cell growth medium, culture at 37 ℃ and 5% CO2 with shaking at 120 rpm; count viable cells and passage every 2-3 days, maintaining a cell concentration of 0.8-1.2 × 10⁻⁶ cells / day. 6 Cells / mL, ensuring a viable cell rate >95%.

[0109] (2) Preparation of virus inoculation tubes: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, and adjust the cell concentration to 3×10⁻⁶. 6 The cells were aliquoted into centrifuge tubes, with 10 mL of cell suspension and 100 μL of 1% triple antibody solution added to each tube. TPCK trypsin at a final concentration of 20 mg / L was added to the inoculation centrifuge tubes and mixed thoroughly for later use.

[0110] (3) Virus inoculation and culture: Take out the clinical sample, freeze and thaw it once, and mix it thoroughly on a shaker; add 0.5 mL of the treated sample to each of the prepared virus inoculation tubes and mix gently. Place the inoculated centrifuge tubes in an incubator at 34 ℃ and 5% CO2 and culture at 180 rpm for 3 days.

[0111] (4) Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge at 3000 rpm for 10 min and collect the supernatant, which is the first generation of virus solution. Repeat the above operation for multiple generations.

[0112] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0113] Table 4. Isolation efficiency of the method of the present invention against HPIV-3 virus. As shown in Tables 2-4, the CT values ​​of RT-PCR in the samples before and after isolation decreased significantly, with an average decrease ranging from 1.53 to 8.92 cycles, indicating effective viral amplification during culture. HA titration results showed that the HA dilution factor for all successfully isolated strains was ≥4. Statistical analysis of the control results showed that the overall virus isolation rate was 52.17%, with a type 1 HPIV isolation rate of 55.56%, a type 2 HPIV isolation rate of 60.00%, and a type 3 HPIV isolation rate of 44.44%. Furthermore, over 85% of type 1 and type 3 HPIV strains were successfully isolated within two generations, and even the more difficult-to-isolate type 2 HPIV strains could be successfully isolated within 3-5 generations.

[0114] Example 10 The materials used in Example 10 of this invention are sourced from the following sources: 1. Cells: Suspension MDCK cells, provided by Shanghai Berger Medical Technology Co., Ltd.

[0115] 2. Virus: Human parainfluenza virus types 1-3, samples were obtained from pharyngeal swabs of febrile patients and provided by Wuhan Children's Hospital.

[0116] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0117] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0118] 5. Triple Antibiotic Solution: Contains the antibiotics penicillin, streptomycin, and amphotericin B, provided by Shanghai Berger Medical Technology Co., Ltd.

[0119] In this embodiment of the invention, viral isolation and culture were performed on 21 pharyngeal swab samples diagnosed as HPIV-positive (6 cases of HPIV-1, 3 cases of HPIV-2, and 12 cases of HPIV-3). The specific culture method is as follows: (1) Preparation of suspension MDCK cells: Using conical shake flasks containing cell growth medium, culture at 37 ℃ and 5% CO2 with shaking at 120 rpm; count viable cells and passage every 2-3 days, maintaining a cell concentration of 0.8-1.2 × 10⁻⁶ cells / day. 6 Cells / mL, ensuring a viable cell rate >95%.

[0120] (2) Preparation of virus inoculation tubes: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, and adjust the cell concentration to 3×10⁻⁶. 6 The cells were aliquoted into centrifuge tubes, with 10 mL of cell suspension and 100 μL of 1% triple antibody solution added to each tube. TPCK trypsin at a final concentration of 20 mg / L was added to the inoculation centrifuge tubes and mixed thoroughly for later use.

[0121] (3) Virus inoculation and culture: Take out the clinical sample, freeze and thaw it once, and mix it thoroughly on a shaker; add 0.5 mL of the treated sample to each of the prepared virus inoculation tubes and mix gently. Place the inoculated centrifuge tubes in an incubator at 34 ℃ and 5% CO2 and culture at 180 rpm for 3 days.

[0122] (4) Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge at 3000 rpm for 10 min and collect the supernatant, which is the first generation of virus solution. Repeat the above operation for multiple generations.

[0123] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0124] Comparative Example 1 The materials used in Comparative Example 1 of this invention are sourced from the following sources: 1. Cells: Conventional adherent LLC-MK2 cells, provided by Hubei Provincial Center for Disease Control and Prevention.

[0125] 2. Virus: Human parainfluenza virus types 1-3, samples were obtained from pharyngeal swabs of febrile patients and provided by Wuhan No. 1 Hospital.

[0126] 3. Cell growth medium: provided by Shanghai Berger Medical Technology Co., Ltd.

[0127] 4. TPCK pancreatin: provided by Sigma-Aldrich, USA.

[0128] In the comparative example of this invention, 21 pharyngeal swab samples diagnosed as HPIV positive (including 6 cases of HPIV-1, 3 cases of HPIV-2, and 12 cases of HPIV-3) were isolated and cultured using conventional adherent LLC-MK2 cells. The specific culture method is as follows: (1) Preparation of adherent LLC-MK2 cells: Resuscitate adherent LLC-MK2 cells and culture them in T25 culture flasks at 37 ℃ and 5% CO2 using cell growth medium (DMEM containing 10% fetal bovine serum). Passage cells are digested with 0.25% trypsin every 2-3 days, with a seeding concentration of 1×10⁶ cells / year. 5 Add 5 mL of cell suspension to each T25 culture flask.

[0129] (2) Preparation of virus inoculation tubes: Adherent cells that have been passaged for 24 hours and are in the logarithmic growth phase are discarded. After washing the cells twice with 1 mL PBS, they can be used for virus inoculation.

[0130] (3) Virus inoculation and culture: The clinical samples were taken out from -80 ℃, frozen and thawed once and shaken to mix. 0.5 mL of the treated sample was added to each inoculation bottle and the bottle was gently shaken to spread the mixture. The inoculation bottles were placed in an incubator at 34 ℃ and 5% CO2 for 2 hours for adsorption, and then 3 mL of DMEM containing 2 mg / L TPCK trypsin was added to each bottle. The bottles were then cultured for another 5 days.

[0131] (4) Virus harvesting and identification: After 5 days of culture, the virus inoculation bottle was placed at -80 ℃ and frozen. After thawing, the culture product was transferred to a 15 mL centrifuge tube and centrifuged (3000 rpm, 10 min) to collect the supernatant, which is the virus solution of the first generation. The above operation was repeated for multiple generations.

[0132] (5) RT-PCR was used to detect the viral CT value and HA titration was performed.

[0133] The statistical results of PCR-CT values ​​of cultures isolated and cultured for one generation from 21 clinical samples in Example 10 and Comparative Example 1 are shown below. Figure 3 Statistical results of PCR-CT values ​​of first-generation cultures of different HPIV virus subtypes from 21 clinical samples. Figure 4 .

[0134] Depend on Figure 3 and Figure 4 It is evident that, based on overall trend analysis and statistical analysis of CT values ​​before and after culture using different methods, the average CT value of the suspension cell culture group was significantly lower than that of the adherent cell culture group. In the culture results for samples targeting different viral subtypes, suspension cells showed a more significant reduction in CT value than adherent cells (difference > 3), with the advantage being particularly pronounced in HPIV-2 and HPIV-3 samples. This indicates that the suspension cell culture system has a higher viral amplification efficiency for HPIV viruses, especially HPIV-2 and HPIV-3 subtypes. This demonstrates that the suspension cell culture system in this invention is generally superior to the traditional adherent cell system in promoting viral amplification, providing important methodological evidence for the clinical isolation, virological research, and potential vaccine development of HPIV.

[0135] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for efficient isolation of human parainfluenza virus based on suspension MDCK cells, characterized by, Includes the following steps: Step 1, Preparation of suspension MDCK cells: Revive the frozen suspension MDCK cells, culture them in an incubator with shaking using cell growth medium, count the viable cells and passage them. Step 2, virus inoculation tube preparation: Take suspension cells in good growth state during the logarithmic growth phase, centrifuge and resuspend in fresh culture medium, adjust the cell concentration and dispense into centrifuge tubes; add TPCK trypsin and triple antibody solution to each tube, mix well and set aside. Step 3, Virus inoculation and culture: After taking the clinical sample out of -80 ℃ and freezing and thawing it once, mix it thoroughly. Add the treated sample to the prepared virus inoculation tube and mix well. Place the inoculated centrifuge tube in an incubator and shake to culture. Step 4, Virus harvesting and identification: After freezing the centrifuge tubes at -80 ℃ and thawing them, centrifuge and collect the supernatant, which is the first-generation virus solution. Repeat the above operation step 3 for passage again to obtain cultured human parainfluenza virus.

2. The method for efficiently separating human parainfluenza virus based on suspended MDCK cells according to claim 1, characterized in that, In step 1, the cell culture temperature is 37 ℃, the CO2 concentration in the incubator is 2~10%, and the shaking speed is 120~180 rpm.

3. The method for efficiently separating human parainfluenza virus based on suspended MDCK cells according to claim 2, characterized in that, In Step 1, live cell counts and passaging should be performed every 2-3 days, maintaining cell concentration at 0.8-1.2 x 10 6 / mL, ensuring a live cell rate of >95%.

4. The method for efficiently separating human parainfluenza virus based on suspended MDCK cells according to any one of claims 1-3, characterized in that, In step 2, the cell concentration was adjusted to 3 x 10 6 / mL to 5 x 10 6 / mL.

5. The method for efficiently isolating human parainfluenza virus based on suspended MDCK cells according to claim 4, characterized in that, In step 2, each inoculation tube contains 10 mL to 30 mL of cell suspension, 100 μL to 300 μL of 1% tri-antibody solution, and 5 to 40 mg / L of TPCK trypsin.

6. A method for efficiently isolating human parainfluenza virus based on suspended MDCK cells according to any one of claims 1-3, characterized in that, In step 3, the amount of clinical sample added is 0.5 mL to 1 mL.

7. The method for efficiently isolating human parainfluenza virus based on suspended MDCK cells according to claim 6, characterized in that, In step 3, the culture temperature is 33 ℃~35 ℃, the CO2 concentration in the incubator is 2~10%, the shaking speed is 180~220 rpm, and the culture time is 3~5 days.

8. A method for efficiently isolating human parainfluenza virus based on suspended MDCK cells according to any one of claims 1-3, characterized in that, In step 4, the centrifugation speed is 2000~3000 rpm and the time is 5~10 min.

9. The method for efficiently isolating human parainfluenza virus based on suspended MDCK cells according to claim 8, characterized in that, In step 4, the virus solution from the first passage is used as the seed virus for the next generation of virus culture, and the cells are passaged 2 to 5 times.

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