PEDV high-affinity monoclonal cell strain and application thereof

Abstract

The application discloses a PEDV high-affinity monoclonal cell strain and application thereof, the cell strain Vero-B6 is preserved in the China General Microbiological Culture Collection Center on June 21, 2023, and the strain preservation number is CGMCC NO.45622.The application screens a Vero cell susceptible to PEDV, and the related characteristics of the screened Vero-B6 cell line are identified in series, and the results show that the Vero-B6 cell is passed to 25 generations, the cells of each generation can be well adhered, the cell passage is stable, after inoculation of PEDV, it is found that the susceptibility to PEDV is also relatively stable, the cell characteristics are kept stable in the passage process, and the cell does not contain bacterial, fungal, mycoplasma and some exogenous virus pollution, and the cell line can be used for laboratory pathogen and vaccine related research.

Description

Technical Field

[0001] This invention belongs to the field of bioimmunotechnology, specifically relating to a PEDV highly tropism monoclonal cell line and its applications. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) was first discovered in 1978. A variant strain emerged in China in 2010, subsequently causing a widespread pandemic in Asia and other European countries, resulting in severe economic losses to the global pig farming industry. PEDV can infect pigs of all ages, with vomiting and diarrhea being the primary clinical symptoms. Severe cases can lead to death, with the highest mortality rate observed in piglets under 10 days old. Currently, there are no effective drugs against PEDV; therefore, prevention and control primarily rely on establishing robust biosecurity measures and clinical detection of the pathogen.

[0003] While there has been some progress in the development of vaccines for Porcine Epidemic Diarrhea (PEDV), the emergence of PEDV variants has rendered vaccines ineffective in protecting pigs from infection. Developing an effective PEDV vaccine is crucial for the prevention and control of this disease. Currently, the Vero cell line is the primary cell line used in laboratories to isolate and culture PEDV. However, PEDV faces challenges in in vitro cell culture, including poor adaptability, unstable viral passage, and low viral titers, which severely hinder research on PEDV and the development of vaccines and other biological products.

[0004] This study aims to screen Vero cells susceptible to PEDV through cell clonal screening, and to identify cell lines that can be stably passaged and proliferate stably with PEDV through systematic identification, laying the foundation for research on PEDV pathogens and vaccines. Summary of the Invention

[0005] The purpose of this invention is to provide a PEDV highly tropism monoclonal cell line and its applications.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A cell line susceptible to PEDV, Vero-B6, classified as green monkey kidney cell, was deposited on June 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.45622. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The application of the aforementioned cell line Vero-B6 in PEDV pathogen-related research or vaccine research.

[0009] Beneficial effects of the present invention

[0010] This invention screened a Vero cell line susceptible to PEDV and performed a series of characterizations on the selected Vero-B6 cell line, including cell line morphology, cell stability (studies on PEDV proliferation stability and cell passaging stability), and identification of exogenous microorganisms. Vero-B6 cells were passaged to 25 generations, with each generation showing good cell adhesion and stable passaging. After PEDV inoculation, susceptibility to PEDV remained relatively stable, indicating that the cell characteristics remained stable during passage. To rule out exogenous microbial contamination, this study used bacterial and fungal detection methods, mycoplasma detection kits, and RT-PCR methods to detect bacteria, fungi, mycoplasma, PEDV, PRRSV, and PoRV. All results were negative, indicating that the cells are free of bacterial, fungal, mycoplasma, and certain exogenous viral contamination. This cell line can be used for laboratory research on pathogens and vaccines. Attached Figure Description

[0011] Figure 1 For IFA testing of PEDV-infected Vero monoclonal cells;

[0012] Wherein, A: negative control; B: Vero cells not selected for clone screening; C: Vero-B6 cells susceptible to PEDV clone; D: B10 cells; E: E7 cells; F: E9 cells; G: F3 cells; H: F8 cells.

[0013] Figure 2 The expression levels of PEDV N protein in Vero blast cells and Vero-B6 cells were detected by Western blotting at different time points.

[0014] Figure 3 The growth curves of PEDV in Vero-B6 and Vero cells are shown.

[0015] Figure 4 The RNA content of PEDV in Vero-B6 and Vero cells at different time points after PEDV infection.

[0016] Figure 5 Cell morphology of Vero mother cell line and clone Vero-B6 cell before and after inoculation with the virus;

[0017] Cells swell and become round; at 18h and 24h of viral infection, both Vero blastoblasts and Vero-B6 cells showed vacuoles and syncytia, but Vero-B6 cells showed more syncytia than Vero blastoblasts; at 36h of viral infection, a large number of syncytia fused to form vacuoles in Vero blastoblasts, but cell shedding was less common, while in contrast, Vero-B6 cells showed cell shedding and most cells exhibited filamentous characteristics.

[0018] Figure 6 To assess the stability of the Vero-B6 cell line against PEDV susceptibility;

[0019] Among them, A: F5 generation; B: F10 generation; C: F15 generation; D: F20 generation.

[0020] Figure 7 The results of mycoplasma detection in Vero-B6 cells;

[0021] Wherein, A: negative control; B: positive control; C: Vero-B6 cell culture supernatant.

[0022] Figure 8 PEDV detection for Vero-B6 cells at different passages.

[0023] Wherein, M: DL2000 marker; N: negative control; lane 1: positive control; lane 2: 5th generation Vero-B6 cells; lane 3: 10th generation Vero-B6 cells; lane 4: 15th generation Vero-B6 cells; lane 5: 20th generation Vero-B6 cells; lane 6: 25th generation Vero-B6 cells.

[0024] Figure 9 PRRSV detection for Vero-B6 cells at different passages.

[0025] Wherein, M: DL2000 marker; N: negative control; lane 1: positive control; lane 2: 5th generation Vero-B6 cells; lane 3: 10th generation Vero-B6 cells; lane 4: 15th generation Vero-B6 cells; lane 5: 20th generation Vero-B6 cells; lane 6: 25th generation Vero-B6 cells.

[0026] Figure 10 PoRV detection for Vero-B6 cells at different passages;

[0027] Wherein, M: DL2000 marker; N: negative control; lane 1: positive control; lane 2: 5th generation Vero-B6 cells; lane 3: 10th generation Vero-B6 cells; lane 4: 15th generation Vero-B6 cells; lane 5: 20th generation Vero-B6 cells; lane 6: 25th generation Vero-B6 cells. Detailed Implementation

[0028] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and these embodiments, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the invention.

[0029] Example 1

[0030] 1. Materials

[0031] 1.1 Cells and Virus Strains

[0032] Vero cell lines and PEDV-MS virus strains were preserved in the laboratory.

[0033] 1.2 Main Instruments and Reagents

[0034] (1) Instruments: Eppendorf PCR instrument, ABI 7500 real-time PCR instrument, Cytiva gel electrophoresis Bio-Rad PCR instrument, real-time fluorescence microscope, Tanon 5200 chemiluminescence imaging instrument.

[0035] (2) Reagents: Calf serum was purchased from Lonsera, FITC goat anti-mouse IgG was purchased from Proteintech, DAPI 10ug / mL was purchased from Bioshard, rapid gel kit was purchased from Yamei Biotechnology Co., Ltd., one-step rapid mycoplasma detection kit was purchased from Yisheng Biotechnology Co., Ltd., DMEM was purchased from Corning, 0.2% trypsin was purchased from Gibco, TMB chromogenic solution was purchased from Yamei Biotechnology Co., Ltd., and anti-PEDV-N monoclonal antibody was stored in this laboratory.

[0036] 2 methods

[0037] 2.1 Vero cell cloning

[0038] Vero cells were resuscitated and cultured into a monolayer in cell culture flasks. The cells were digested with trypsin, and after digestion, 2 mL of DMEM nutrient solution containing 10% (v / v) serum was added to the cells and they were pipetted into single cells. After a 10-fold dilution, 10 μL of the solution was added to a hemocytometer for counting. The cell suspension was then diluted to 1 cell / 100 μL and 100 μL was added to each well of a 96-well plate. The cells were cultured at 37°C and 5% CO2 for about 15 days. The cell wells were observed daily, and the number of cells that grew from single cells was recorded and expanded. Single clones were diluted into single cells again following the above steps and seeded onto new plates for expansion culture. After the cells reached a confluent monolayer, they were infected with PEDV. Indirect immunofluorescence (IFA) was performed 24 hours later to identify cell lines susceptible to PEDV. Then, further subcloning was performed. After three subclonings, PEDV was inoculated and indirect immunofluorescence (IFA) was used to detect the susceptibility of the monoclonal cell lines to PEDV. Monoclonal Vero cell lines sensitive to PEDV proliferation were selected and passaged to the third generation (referred to as P1 generation). Some of these cells were cryopreserved, and some were passaged again for PEDV susceptibility identification.

[0039] 2.2 PEDV-susceptible cell screening

[0040] 2.2.1 Indirect Immunofluorescence Assay (IFA)

[0041] (1) Sample preparation: Different clones of Vero cells were seeded into 96-well plates. After the cells reached a confluent monolayer, 0.01 MOI PEDV-MS (10⁻⁶ cells / well) were seeded into each well. 4.8 TCID 50 Add 4% (ml / 100ml) serum to maintenance medium (containing 2% trypsin), incubate at 37℃ and 5% CO2 for 24h, and then perform IFA test;

[0042] (2) Fixation: Discard the maintenance solution for different Vero clones, wash three times with PBST for 10 minutes each time, add 50 μL of pre-cooled anhydrous ethanol, and fix for 15 minutes; after fixation, discard the fixative, wash three times with PBST for 5-10 minutes each time.

[0043] (3) Permeability: Add 50 μL of 0.1% (v / v) Triton X-100 to each well and let it sit at room temperature for 10 min;

[0044] (4) Blocking: Discard the permeation solution, add 100 μL PBST to each well to wash the cells 3 times, add 3% (g / 100ml) BSA, 50 μL / well, and block at room temperature for 1 h;

[0045] (5) Primary antibody incubation: Add 50 μL of PEDV N monoclonal antibody (diluted with 3% (g / 100ml) BSA at a ratio of 1:200) to each well and incubate overnight at 4°C;

[0046] (6) Secondary antibody incubation: Wash cells 3 times with PBST, add 50 μL of FITC goat anti-mouse secondary antibody (diluted with 3% (g / 100ml) BSA at 1:200) to each well, and incubate at room temperature in the dark for 2 h;

[0047] (7) DAPI: Wash cells three times with PBST, add 50 μL of 5% (g / 100ml) DAPI to each well and stain the nuclei for 10 min; after staining the nuclei, wash the cells three times with PBST and observe them under a microscope.

[0048] 2.2.2 Western Blot Identification

[0049] (1) Sample preparation: Vero cells and susceptible monoclonal cells were seeded into a monolayer in a 24-well plate and inoculated with 0.01 MOI PEDV-MS virus. After discarding the nutrient solution at 12, 18, 24 and 36 h, the samples were washed three times with PBS and lysis buffer was added to collect protein samples. The collected samples were then boiled. The treated samples were stored at -20℃.

[0050] (2) Electrophoresis: Prepare a 10% gel and centrifuge the frozen sample at 4℃ and 12000rpm for 10min. Add 10-15μL of sample to the sample well and perform electrophoresis at 80V for 30min. After the sample enters the separating gel, increase the voltage to 120V.

[0051] (3) Transfer: After rinsing with transfer solution, place the sponge, filter paper, membrane, gel, filter paper, and sponge into the transfer clamp in the following order, and transfer at 120V for 1.5h.

[0052] (4) Sealing: Take out the transferred film and seal it with 10% skim milk at room temperature for 1 hour.

[0053] (5) Antibody binding: The blocked membrane was washed with PBST 3-5 times for 10 min each time, and then placed in mouse anti-PEDV-N antibody and mouse anti-GAPDH respectively, and incubated overnight at 4°C. The membrane incubated overnight was washed with PBST 5 times, and then incubated in 1:5000 goat anti-mouse IgG-HRP at room temperature for 1 h.

[0054] (6) Development: Wash with PBST 3-5 times, add Yamei developer, and develop using the Tanon chemical development system.

[0055] 2.2.3 Virus TCID 50 Measurement and its growth curve

[0056] (1) Sample preparation: Vero cells and PEDV-susceptible monoclonal cells were seeded in 24-well plates. After 24 hours, the cells were seeded into 0.01 MOI PEDV-MS. The supernatant was collected at 12 hours, 18 hours, 24 hours and 36 hours after infection. The samples were stored at -20°C.

[0057] (2) TCID 50 Detection: Samples collected at different times were serially diluted with virus maintenance solution containing 4% trypsin, and the dilution was taken as 10. -1 -10 -7 Samples were inoculated into 96-well plates confluent with Vero monolayer cells at a rate of 0.1 ml / well, with 6 wells inoculated for each dilution. Uninoculated cells served as a negative control. The plates were incubated at 37°C and 5% CO2 for 48 h. The number of diseased wells at different dilutions was recorded, and the viral TCID was calculated using the Karber method. 50 Repeat the experiment three times and plot the results to create a virus growth curve.

[0058] 2.2.4 Real-time PCR detection of viral proliferation

[0059] Appropriate amounts of Vero cells and susceptible monoclonal Vero cells identified by IFA were seeded into 24-well plates. After both cell lines had grown into a confluent monolayer, 0.01 MOI PEDV-MS virus solution (10⁻⁶) was inoculated into each well. 4.8 TCID 50 Cells were added to maintenance medium and cell supernatants were collected at 12, 18, 24, and 36 h. Nucleic acid from TGEV and PDCoV viruses was extracted using the OMEGA RNA Extraction Kit according to the manufacturer's instructions. The RNA was washed with 40 μL of RNase-free water. The extracted RNA was reverse transcribed using HiScipt Reverse Transcripatase. The reaction mixture consisted of 2 μL of 5x HiScipt Reverse Transcripatase Mix and 8 μL of RNA. The reverse transcription PCR program was: 37℃ for 15 min, 85℃ for 5 s, and cDNA was stored at -20℃ after reverse transcription. The viral supernatant was quantitatively analyzed using PEDV fluorescent RT-PCR. The fluorescent quantitative RT-PCR reaction conditions were: contamination elimination 25℃ for 10 min; pre-denaturation 95℃ for 30 s; denaturation 95℃ for 10 s; annealing and extension 60℃ for 34 s, for 40 cycles. Primers, probes, and reaction systems are shown in Tables 1 and 2.

[0060] Table 1. PEDV primer and probe sequences

[0061]

[0062] Table 2 PEDV Real-Time RT-PCR Reaction System

[0063]

[0064] 2.3 Vero-B6 cell identification

[0065] To investigate the stability of PEDV-susceptible Vero-B6 cells, the following cell characteristics were identified.

[0066] 2.3.1 Vero-B6 cell morphology study

[0067] Equal amounts of Vero cells and Vero-B6 cells were seeded into 24-well plates. Once the cells reached a confluent monolayer, morphological differences between the two cell types were observed. Each cell type was then seeded with 0.01 MOI PEDV-MS virus (102). 4.8 TCID 50 Cell morphology changes were observed at 12, 18, 24 and 36 h after inoculation ( / mL).

[0068] 2.3.2 Vero-B6 cell passage, cryopreservation and resuscitation

[0069] After thawing the frozen Vero-B6 cells, they were cultured into a monolayer in cell culture flasks, digested with 0.2% trypsin, and divided into flasks at a 1:2 ratio. Culture medium was added, and the cells were cultured at 37°C and 5% CO2 for 24-48 hours. The cells were then passaged to passage 25. During passage, cells from passages 5, 10, 15, 20, and 25 were cryopreserved. One to two cell samples were taken every week for thawing, and cell growth and activity were observed.

[0070] 2.3.3 Stability of Vero-B6 cells to PEDV proliferation

[0071] The cells selected from the above clones were passaged, and cells from passages 5, 10, 15, and 20 were seeded in 96-well plates. After the cells reached a confluent monolayer, they were inoculated with 0.01 MOI of PEDV-MS virus solution and cultured for 24 hours. IFA was then performed to determine whether the susceptibility of Vero-B6 cells to PEDV was stable.

[0072] 2.3.4 Detection of other exogenous microorganisms in Vero-B6 cells

[0073] 2.3.4.1 Detection of bacteria and fungi in Vero-B6 cells

[0074] After culturing for 24 hours, the cells were digested with trypsin and then a suitable amount of nutrient solution was added to prepare a cell suspension. The cell suspension was inoculated into ordinary broth medium for enrichment, and then the suspension was added to ordinary broth medium and modified Martin medium respectively. The broth medium was placed at 37°C and the modified Martin medium was placed at 25°C for 3-7 days, and the colony growth in the medium was observed.

[0075] 2.3.4.2 Vero-B6 cell mycoplasma detection

[0076] The presence of mycoplasma in cells was detected using a one-step rapid mycoplasma detection kit purchased from Yisheng Biotechnology Co., Ltd. The specific procedure was as follows: The kit was removed from -20℃ and set aside. The reaction system is shown in Table 3. A PCR instrument was used for the reaction, with the reaction conditions being 30℃ for 5 min and 63℃ for 60 min. After the reaction, the reaction tubes were removed and placed at room temperature. The reaction results were observed under good lighting conditions with a white paper background. The negative control was blue-purple, and the positive control was sky blue. The sample color was compared with the positive and negative controls to determine whether the cells contained mycoplasma contamination.

[0077] Table 3 Mycoplasma Detection Reaction System

[0078]

[0079] 2.3.5 Vero-B6 cell extracellular virus detection

[0080] To detect whether there is exogenous viral contamination in the cells, Vero-B6 cells were seeded in 24-well plates. After the cells grew into a monolayer, they were collected, RNA was extracted, and reverse transcription was performed using HiScipt Reverse Transcripatase (see Chapter 2, 2.2.1). PEDV, PRRSV, and PoRV specific primers (Table 4) were used to perform conventional RT-PCR reactions to detect different viruses. The reaction system and reaction conditions are shown in Tables 5 and 6.

[0081] Table 4 Primer sequences for PEDV, PRRSV, and PoRV

[0082]

[0083] Table 5 PCR reaction system

[0084]

[0085] Table 6. PCR reaction procedure for exogenous virus detection

[0086]

[0087] 3 Results

[0088] 3.1 Screening of PEDV-susceptible cells

[0089] 3.1.1 Cell Cloning and PEDV IFA Detection

[0090] Vero cells were cloned, resulting in six cloned cell lines, named B6, B10, E7, E9, F3, and F8. IFA experiments were performed, and the results showed ( Figure 1 48 hours after infection with the virus, the green fluorescent staining of B6 cells was significantly higher than that of other clones, indicating that this cell type is most susceptible to PEDV.

[0091] 3.1.2 Western Blot detection of N protein expression level

[0092] To further identify the susceptibility of B6 cells to PEDV, Western blotting was used to detect the expression level of N protein at different time points after Vero-B6 infection with the virus. The results are as follows: Figure 2 As shown, the expression level of N protein in Vero-B6 cells was significantly higher than that in Vero cells at 12, 18, 24, and 36 hours after infection. This result is consistent with the IFA results, indicating that Vero-B6 cells are more susceptible to PEDV than Vero cells.

[0093] 3.1.3 Virus TCID 50 Measurement and its growth curve

[0094] To investigate the differences in viral proliferation between Vero blast cells and Vero-B6 cells after viral infection, culture supernatants were collected at 12h, 18h, 24h, and 36h after cell infection with 0.01 MOI PEDV-MS to determine TCID. 50 The value was repeated 3 times. Then, a virus growth curve was plotted, and the results were as follows. Figure 3 As shown, at all stages of PEDV proliferation, the viral titer in Vero-B6 cells was higher than that in Vero cells, and the TCID levels of Vero-B6 cells and Vero cells were also higher at 24h and 36h. 50 The differences were even more significant. The results showed that PEDV had a stronger tropism for Vero-B6 cells and a higher viral replication titer.

[0095] 3.1.4 Real-time PCR

[0096] To further determine the differences in viral proliferation after cell infection, the amount of viral RNA in the cell supernatant of Vero and Vero-B6 cells at different time points after PEDV infection was measured by PEDV quantitative real-time PCR. Figure 4The results showed that the viral RNA content in Vero-B6 cells was higher than that in Vero cells at all time points after PEDV infection. Furthermore, the difference in viral RNA content in the supernatant of the two cell lines gradually increased with the duration of infection. The most significant difference in viral RNA content between Vero-B6 cells and Vero cells was observed at 36 hours after viral infection, indicating that PEDV has a stronger proliferation capacity in Vero-B6 cells.

[0097] 3.2 Identification of Vero-B6 cells

[0098] 3.2.1 Cell morphology study

[0099] After culturing Vero blastoblasts and Vero-B6 cells for 24 days, morphological differences were observed under a microscope, such as... Figure 5 As shown, there was no significant morphological difference between Vero-B6 cells and Vero blast cells. After the cells reached a monolayer, they were inoculated with PEDV. At 12 h of viral infection, both Vero blast cells and Vero-B6 cells exhibited the viral infection morphology, i.e., the infected cells were swollen and rounded. At 18 h and 24 h of viral infection, both Vero blast cells and Vero-B6 cells showed vacuoles and syncytiomes, but Vero-B6 cells showed more syncytiomes than Vero blast cells. At 36 h of viral infection, a large number of syncytiomes fused to form vacuoles in Vero blast cells, but cell detachment was relatively rare. In contrast, Vero-B6 cells showed cell detachment and most cells exhibited filamentous characteristics.

[0100] 3.2.2 Clonal cell passage, cryopreservation and thawing

[0101] Vero-B6 cells were passaged to 25 generations, and cells in each generation were able to adhere and grow, with consistent cell morphology. Cells from generations 5, 10, 15, 20, and 25 were cryopreserved at -196°C in liquid nitrogen and then thawed, and the cells grew well without any abnormalities.

[0102] 3.2.3 Stability of Vero-B6 to PEDV proliferation

[0103] Vero-B6 at passages 5, 10, 15, and 20 were inoculated with PEDV and subjected to IFA. Results ( Figure 6 The results showed that the fluorescence levels in cells of different passages were comparable, with no significant difference, indicating that the susceptibility of Vero-B6 cells to PEDV did not change with passage number.

[0104] 3.2.4 Detection of other exogenous microorganisms in Vero-B6 cells

[0105] Different methods were used to detect bacteria, fungi, and mycoplasma in Vero-B6 cells.

[0106] 3.2.4.1 Detection of bacteria and fungi in Vero-B6 cells

[0107] After 24 hours of culture, cells were digested with trypsin, and culture medium was added to stop the digestion. Then, an appropriate amount of culture medium was added to prepare a cell suspension. The cell suspension was inoculated into ordinary broth medium and modified Martin medium, respectively, and cultured at 37℃ and 25℃, respectively. The results showed that both were negative, indicating that Vero-B6 cells were free from bacterial and fungal contamination.

[0108] 3.2.4.2 Vero-B6 cell mycoplasma detection

[0109] Vero-B6 cell supernatant was tested for mycoplasma, and the results were ( Figure 7 The results showed that the culture supernatant of Vero-B6 cells was negative, indicating that Vero-B6 cells were not contaminated with mycoplasma.

[0110] 3.2.5 Vero-B6 exogenous virus detection

[0111] Vero-B6 cells from passages 5, 10, 15, 20, and 25 were cultured into monolayers, and cell samples were collected for RNA extraction and detection of exogenous PEDV, PRRSV, and PoRV viruses.

[0112] 3.2.5.1 PEDV test results

[0113] PEDV results were detected using RT-PCR. Figure 8 The results showed that Vero-B6 cells from passages 5, 10, 15, 20 and 25 did not contain PEDV.

[0114] 3.2.5.3 PRRSV Detection Results

[0115] RT-PCR detection of PRRSV, results ( Figure 9 The results showed that Vero-B6 cells from passages 5, 10, 15, 20, and 25 did not contain PRRSV.

[0116] 3.2.5.4 PoRV test results

[0117] The PoRV results are as follows: Figure 10 The results showed that Vero-B6 cells from passages 5, 10, 15, 20, and 25 did not contain PoRV.

[0118] 4 Discussion

[0119] PEDV is one of the major gastrointestinal viruses causing diarrhea in pigs. It first appeared in the UK and Belgium in the early 1970s, and subsequently spread to other European countries. Before the emergence of a new variant in China in 2010, vaccines related to the traditional CV777 strain were effective in controlling PEDV outbreaks. Due to the use of vaccines, PEDV was sporadic and did not attract much attention globally. After the emergence of a new PEDV variant between 2010 and 2012 (Li et al., 2012b), inactivated vaccines against the traditional CV777 strain were no longer effective in reducing piglet mortality (Wang et al., 2013). This necessitates research into the pathogen and the development of vaccines or antiviral agents against the variant. The first step in pathogen research is virus isolation, the most common method being cell culture. Since PEDV was discovered in China in 1984, its in vitro isolation and culture has been a research challenge.

[0120] Studies on the screening and preparation of virus-sensitive cells have been conducted both domestically and internationally. Some scholars have used limiting dilution methods (Zheng Pei'e, 1999) to screen cell lines susceptible to PEDV or other viruses. For example, Chen Nianqu (Chen Nianqu, 2009) used limiting dilution to screen the PK-15-B1 cell line susceptible to PCV2, which produced high titers of PCV2. Lu Quanwei (Lu Quanwei, 2013) also screened the PK15-G6 cell line susceptible to PCV2, and the susceptibility of this cell line to PCV2 was relatively stable and did not change with passage number. Foreign scholars Qingzhan Zhang (Zhang et al., 2018) screened the IPEC-DQ cell line susceptible to PEDV and successfully used it for subsequent PEDV mechanism research. From the research of the above scholars, it can be hypothesized that different cells in the same Vero cell line have different susceptibility to PEDV. Following the screening method described above, this study used limiting dilution to screen for PEDV-susceptible monoclonal cell lines. IFA was used to screen for Vero cell lines with the highest positive rate after PEDV infection. After three subcloning processes, a cell line, Vero-B6, susceptible to PEDV was finally selected. After inoculation with PEDV-MS, Western Blot, Real-time PCR, and TCID50 experiments were performed to further confirm the high sensitivity of this cell line to PEDV.

[0121] In vitro cell culture artificially provides cells with an environment that simulates the in vivo environment. This culture environment differs somewhat from the dynamic in vivo environment. When cells grow to a certain density, they need to be separated into different flasks or replaced with new nutrient solutions to continue growing; otherwise, the cells will die. This process is called passage. Each passage has a certain impact on the state and proliferation level of the cells. Cell growth and passage is a developmental process, which leads to different cells having different nutritional requirements. Currently, the definition of a cloned cell line is: a cell population derived from a single cell selected through different screening methods from a biologically identified cell line.

[0122] In recent years, there has been no unified standard for what criteria a cell line must meet to be considered a biologically validated cell line. However, establishing a cell line should meet the following requirements: the source of the cell or tissue donor must be clearly stated, such as whether the cells come from a human or animal, and from which organ or tissue; a series of biological tests should be performed on the cells to understand their basic and specific biological characteristics, such as cell morphology, structure, and mitotic index; specific characteristics refer to the fact that some tumor cells should prove that the cells originated from the original tumor tissue and retain their inherent characteristics; different cells should have their own suitable growth environment and culture conditions, and the cell culture conditions, such as serum, culture medium, suitable temperature, and pH, should be clearly stated. In addition, any exogenous microbial contamination during cell culture, such as mycoplasma, bacteria, fungi, or viruses, will lead to a deterioration in cell condition and even cell death; cells containing exogenous contamination will cause virus isolation failure or a decrease in virus titer. Although commercially available culture media, serums, and culture flasks undergo sterility and mycoplasma testing before being sold, sources of contamination are ubiquitous in daily operations. For example, incomplete sterilization of biocabinets, inadequate hand disinfection during experiments, and bacterial or fungal contamination in incubators can all lead to exogenous contamination of cells. Therefore, it is essential to detect exogenous contamination in cells.

[0123] Therefore, this study conducted a series of characterizations on the selected Vero-B6 cell line, including cell line morphology, cell stability (stability against PEDV proliferation and passaging), and identification of exogenous microorganisms. Vero-B6 cells were passaged to 25 generations, with each generation showing good cell adhesion and stable passaging. After PEDV inoculation, susceptibility to PEDV remained relatively stable, indicating that the cell characteristics remained stable during passage. To rule out exogenous microbial contamination, this study used bacterial and fungal detection methods, mycoplasma detection kits, and RT-PCR methods to detect bacteria, fungi, mycoplasma, PEDV, PRRSV, and PoRV. All results were negative, indicating that the cells are free of bacterial, fungal, mycoplasma, and certain exogenous viral contamination. This cell line can be used for laboratory research on pathogens and vaccines.

Claims

1. A cell line Vero-B6 susceptible to PEDV virus, which was deposited on June 21, 2023 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.45622, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The application of the Vero-B6 cell line as described in claim 1 in PEDV vaccine research.

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