Method for identifying PEDV chimeric virus attenuation based on reverse genetic manipulation and application of screening potential attenuated vaccine

Abstract

The application discloses a PEDV chimeric virus attenuation identification method based on reverse genetic operation and application of screening potential attenuated vaccine, and the PEDV chimeric virus CHM2013-SP BJ , CHM2013- BJ , CHM2013-SP BJ -ORF3 CHM , CHM2013-SP BJ -E CHM , CHM2013-SP BJ -M CHM , CHM2013-SP BJ -N CHM and CHM2013-(S+ORF3) BJ are constructed and rescued, loss-of-function test and gain-of-function test are carried out, and the attenuated chimeric virus CHM2013-SP BJ -ORF3 CHM and CHM2013-SP BJ -M CHM are obtained, and it is disclosed that the S gene of the PEDV variant BJ2011C promotes virus pathogenicity in cooperation with the ORF3, E and M genes, and the ORF3 gene is the most critical.

Description

Technical Field

[0001] This invention relates to the technical field of viral genetic engineering, and in particular to a method for identifying attenuated PEDV chimeric viruses based on reverse genetics and its application in screening attenuated vaccines. Background Technology

[0002] The PEDV variant is more pathogenic, and infected piglets exhibit watery diarrhea, anorexia, and dehydration, with a mortality rate of up to 100% in newborn piglets. Compared with the classic strain, the variant has four hypervariable regions in its genome: V1, V2, V3, and V4. V1 is located at the N-terminus of ORF1a, V2 is located in the S1 region of the S gene, V3 includes the 3' end of the S gene and the 5' end of the ORF3 gene, and V4 is located in the N gene (see Sun M, Ma J, Wang Y, et al. Genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in Asia[J].J Clin Microbiol,2015,53(5):1484-92).

[0003] PEDVS genes exhibit genetic diversity. Compared to classic strains, PEDV variants show characteristic base insertions and deletions in the S gene. The laboratory-constructed and rescued PEDV chimeric virus CHM2013-SP... BJ It is pathogenic and can colonize the intestines, CHM2013-S BJ The loss of pathogenicity and inability to colonize the intestine indicate that the S gene mutation is a necessary but not sufficient condition for enhanced pathogenicity of PEDV variants, with structural proteins synergistically promoting viral pathogenicity and intestinal colonization. The relationship between the S gene mutation in PEDV variants and their enhanced pathogenicity remains unknown. Summary of the Invention

[0004] The purpose of this invention is to provide a method for identifying attenuated PEDV chimeric viruses based on reverse genetics and its application in screening potential attenuated vaccines, thereby screening out potential attenuated vaccine candidate strains with low pathogenicity that can colonize the intestine.

[0005] To achieve the above objectives, this invention provides a method for attenuating PEDV chimeric viruses based on reverse genetics, comprising the following steps:

[0006] S1. First, using CHM2013 as the backbone, construct and rescue the chimeric virus CHM2013-SP, which has replaced the coding region of the BJ2011C structural protein and the S protein, respectively. BJ and CHM2013-S BJ Then, using CHM2013-SP BJ Using this as a backbone, we constructed and rescued the chimeric virus CHM2013-SP, which had its structural proteins replaced by CHM2013. BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM and CHM2013-SP BJ -N CHM We conducted loss-of-function experiments to explore the molecular basis of how the S protein and other structural proteins in the PEDV mutant strain BJ2011C synergistically promote pathogenicity.

[0007] S2. Using 2-day-old SPF piglets as a model, administer 5×10 ml orally. 5 TCID 50 Chimeric virus (CHM2013-SP) BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM and CHM2013-SP BJ -N CHM Loss-of-function tests were conducted. The pathogenicity of the chimeric virus to piglets was analyzed based on clinical findings, daily viral shedding, intestinal pathological changes, and intestinal colonization. Results showed that, compared to CHM2013-SP... BJ In comparison, CHM2013-SP BJ -N CHM Pathogenicity has not been reduced; CHM2013-SP BJ -M CHM and CHM2013-SP BJ -E CHM The pathogenicity of CHM2013-SP has decreased; BJ -ORF3 CHM The virus lost its pathogenicity, indicating that the S gene of the PEDV variant, along with the structural proteins ORF3, E, and M, promotes viral pathogenicity, with the S gene working in conjunction with ORF3 being the most crucial in promoting pathogenicity.

[0008] S3. Based on the results of the loss-of-function test, using CHM2013-S BJ Using this as a framework, construct and rescue the chimeric virus CHM2013-(S+ORF3) that replaces BJ2011CORF3. BJ We will conduct gain-of-function assays to further explore the molecular basis of the synergistic pathogenicity promotion by the S protein and ORF3.

[0009] S4. Using 2-day-old SPF piglets as a model, administer 5×10 ml orally. 5 TCID 50 The chimeric virus CHM2013-S BJ And CHM2013-(S+ORF3) BJ The pathogenicity of the chimeric virus in piglets was analyzed based on clinical findings, daily viral shedding, intestinal pathological changes, and intestinal colonization. Further investigation was conducted to determine whether the S gene and structural protein ORF3 of the highly virulent strain BJ2011C synergistically promote viral pathogenicity. Results showed that CHM2013-(S+ORF3) BJ Its pathogenicity is higher than that of CHM2013-S BJ This study revealed that the co-structural protein ORF3 of the PEDV mutant strain BJ2011C S gene promotes viral pathogenicity.

[0010] Furthermore, it also includes:

[0011] S5. After piglet challenge, the survival status of piglets was recorded daily. Piglet weight was measured and recorded before challenge, at death, and at the end of the animal experiment. The severity of diarrhea and mental state of the piglets were carefully observed and scored according to the scoring criteria. By comparing survival curves, average weight gain, clinical scores, and diarrhea scores, the results were compared with CHM2013-SP in the loss-of-function test. BJ In comparison, CHM2013-SP BJ -N CHM It is lethal and highly pathogenic; CHM2013-SP BJ -M CHM and CHM2013-SP BJ -E CHM The pathogenicity has decreased, and infected piglets show diarrhea symptoms; CHM2013-SP BJ -ORF3 CHM It lost its pathogenicity, and infected piglets showed normal clinical symptoms. In the gain-of-function test, compared to CHM2013-S... BJ CHM2013-(S+ORF3) BJ It is more pathogenic to piglets, and infected piglets show earlier onset of illness and more severe diarrhea symptoms.

[0012] S6. After viral challenge, anal swabs were collected daily from piglets, and RT-PCR was used to detect viral shedding in infected piglets. Loss-of-function assay results indicated that CHM2013-SP BJ -ORF3 CHM Infected piglets can end viral shedding earlier, CHM2013-SP BJ -M CHM It delays the time of viral shedding in piglets. Gain of function test results indicate that the chimeric virus CHM2013-S BJ And CHM2013-(S+ORF3) BJ Its ability to reproduce and detoxify in the intestines is relatively weak.

[0013] S7. Observe the gross and histopathological changes in piglets, and perform the loss-of-function test (CHM2013-SP). BJ With CHM2013-SP BJ -N CHM It causes the most severe damage to the intestines, CHM2013-SP BJ -M CHM and CHM2013-SP BJ -E CHM Thinning of the intestinal wall in piglets is the second most damaging factor to the intestines, CHM2013-SP BJ -ORF3 CHM No intestinal lesions were observed in the CHM2013-(S+ORF3) group and the MOCK group piglets. In the functional gain test, CHM2013-(S+ORF3) BJ Compared to CHM2013-S BJ It can cause more serious gross pathological changes.

[0014] S8. Detection of the PEDV genome in intestinal tissue showed that all chimeric viruses were detectable in the intestine during the loss-of-function assay, while the chimeric virus CHM2013-S was not detected in the gain-of-function assay. BJ It cannot colonize the intestine; it is a chimeric virus CHM2013-(S+ORF3). BJ It can colonize the gut, revealing that replacing any structural protein does not alter CHM2013-SP. BJ The ability of viruses to colonize the gut is enhanced, and the structural protein S protein can work in conjunction with ORF3 to promote viral intestinal colonization.

[0015] Furthermore, the interval between steps S1 and S3 also includes:

[0016] Amplification and sequencing of the chimeric virus confirmed that the PEDV chimeric virus had no base mutations. Indirect immunofluorescence identification results showed that syncytial-like green fluorescence could be observed in Vero CCL81 cells, indicating that the chimeric virus was successfully rescued.

[0017] Furthermore, steps S1 and S3 also include the following steps:

[0018] The multi-step growth curve of PEDV chimeric virus on Vero CCL81 cells was determined. Virus was inoculated into 24-well plates containing a monolayer of Vero CCL81 cells at an MOI of 0.01. Viral fluid was collected at different time points post-infection, with three replicates at each time point. The virus was analyzed using TCID50. 50 The viral titer was determined at various time points, and a multi-step growth curve of the chimeric virus was plotted.

[0019] Furthermore, step S7 also includes:

[0020] In the pathogenicity test on piglets, dead piglets were necropsed, and piglets 7 days after challenge were euthanized and necropsed to observe gross pathological changes in the intestines. Intestinal tissue samples were collected and pathological sections were prepared, and the tissue lesions were observed by HE staining.

[0021] Step S8 uses absolute fluorescence quantification and nucleic acid in situ hybridization to detect the PEDV genome in intestinal tissue:

[0022] Intestinal samples were collected from different segments of piglets, and the number of chimeric virus genome copies per unit weight of each intestinal segment was detected using absolute real-time PCR probe method. In situ hybridization with nucleic acid probes was then used to further detect viral colonization in the intestine.

[0023] This invention also provides the chimeric virus CHM2013-SP BJ -ORF3 CHM and CHM2013-SP BJ -M CHM Application as a potential attenuated vaccine candidate strain:

[0024] Immunoprotection test in piglets against CHM2013-SP BJ -ORF3 CHM and CHM2013-SP BJ -M CHM The immunoprotective effect of the chimeric attenuated vaccine candidate strain was evaluated. Results showed that the daily viral load decreased 8 days after immunization, and no viral shedding was detected by RT-PCR 12 days after immunization, indicating the safety of the chimeric attenuated strain. Specific neutralizing antibodies were detected in the serum of immunized piglets, with titers reaching up to 1:84, indicating that the chimeric attenuated strain could induce an immune response in piglets.

[0025] Compared with the prior art, the PEDV chimeric virus attenuation method based on reverse genetics and the application of screening attenuated vaccines according to the present invention have the following advantages:

[0026] The PEDV variant's S protein, along with structural proteins ORF3, E, and M, promotes viral pathogenicity and intestinal colonization, with ORF3 playing the most crucial role. Two chimeric viruses with low pathogenicity and capable of intestinal colonization were screened: CHM2013-SP. BJ -ORF3 CHM and CHM2013-SP BJ -M CHM It is expected to serve as a potential candidate strain for attenuated vaccine, laying a theoretical foundation for the prevention and control of PEDV. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of a chimeric infectious clonal plasmid according to an embodiment of the present invention;

[0028] Figure 2 This is a schematic diagram of cytopathic effect and immunofluorescence identification of a virus rescued according to an embodiment of the present invention;

[0029] Figure 3 This is a schematic diagram illustrating the proliferation dynamics of a chimeric virus in Vero CCL81 cells according to an embodiment of the present invention.

[0030] Figure 4 This is a schematic diagram showing the clinical symptoms of piglets undergoing a loss-of-function test according to an embodiment of the present invention.

[0031] Figure 5 This is a schematic diagram showing the clinical symptoms of piglets in a functional gain test according to an embodiment of the present invention;

[0032] Figure 6 This is a schematic diagram showing the daily viral shedding results of chimeric virus-infected piglets according to an embodiment of the present invention.

[0033] Figure 7 This is a schematic diagram showing the gross pathological changes in the intestine of piglets infected with a chimeric virus according to an embodiment of the present invention.

[0034] Figure 8 This is a schematic diagram showing the histopathological changes in the intestine of piglets infected with a chimeric virus according to an embodiment of the present invention.

[0035] Figure 9 This is a schematic diagram showing the viral load results of chimeric virus infection in different segments of the piglet's intestine according to an embodiment of the present invention.

[0036] Figure 10 This is a schematic diagram showing the results of colonization analysis of a chimeric virus in the intestine of piglets according to an embodiment of the present invention.

[0037] Figure 11 This is a schematic diagram of the daily toxin-shedding copy number in the intestine of piglets according to an embodiment of the present invention;

[0038] Figure 12 This is a schematic diagram illustrating the daily viral shedding of piglets in different infection groups using RT-PCR according to an embodiment of the present invention.

[0039] Figure 13 This is a schematic diagram showing the clinical results of piglets infected with BJ2011C according to an embodiment of the present invention.

[0040] Figure 14 This is a schematic diagram of the enterovirus RNA load in piglets infected with BJ2011C according to an embodiment of the present invention.

[0041] Figure 15 This describes the intestinal pathological changes in piglets infected with BJ2011C according to an embodiment of the present invention.

[0042] Figure 16 This is a schematic diagram showing the antibody titer results in piglet serum according to an embodiment of the present invention. Detailed Implementation

[0043] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0044] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0045] By replacing the structure protein (SP) region of the high-virulence porcine epidemic diarrhea virus (PEDV) strain BJ2011C with the low-virulence PEDV strain CHM2013 as the backbone, a chimeric virus CHM2013-SP was obtained. BJ It is pathogenic to 2-day-old piglets. The chimeric virus CHM2013-S was obtained by replacing the protein of the highly virulent strain BJ2011CS with CHM2013 as the backbone. BJ (Sequence number SEQ ID NO.1) showed attenuated virulence in 2-day-old piglets. Based on these results, further reverse genetics was used to further investigate the virulence of CHM2013-SP. BJ (Sequence SEQ ID NO.2) was used as the parent strain to construct and rescue a chimeric virus that exchanged the structural proteins ORF3, E, M, and N of the low-virulence strain CHM2013: CHM2013-SP BJ -ORF3 CHM(Sequence is SEQ ID NO.3), CHM2013-SP BJ -E CHM (Sequence is SEQ ID NO.4), CHM2013-SP BJ -M CHM (Sequence number SEQ ID NO.5) and CHM2013-SP BJ -N CHM (Sequence number SEQ ID NO.6). Two-day-old SPF piglets were used as the experimental subjects, with a dosage of 5 × 10⁻⁶. 5 TCID 50 The chimeric viruses were challenged via oral inoculation and their pathogenicity was analyzed. Loss-of-function assays showed that all chimeric viruses could colonize the intestine, and compared to the parental strain, CHM2013-SP... BJ -ORF3 CHM Clinical symptoms and intestinal pathological changes were the same as in the MOCK group, and the disease was lost; CHM2013-SP BJ -M CHM and CHM2013-SP BJ -E CHM It can cause diarrhea in piglets, but reduces intestinal pathological changes and weakens virulence; while CHM2013-SP BJ -N CHM The clinical symptoms and intestinal pathological changes were severe, and the pathogenicity was not diminished. The study also used CHM2013-S... BJ The chimeric virus CHM2013-(S+ORF3) was rescued by constructing a skeleton to replace BJ2011CORF3. BJ (Sequence number SEQ ID NO.7). Gain-of-function testing was performed. Results showed that CHM2013-(S+ORF3) BJ The severity of diarrhea and the histopathological changes in the intestines were higher than those in CHM2013-S. BJ Furthermore, it can colonize the intestines, while CHM2013-S BJ It cannot colonize the intestine. The above results indicate that the PEDV mutant S protein, in conjunction with ORF3, E, and M proteins, promotes the pathogenicity of the mutant, with ORF3 playing the most important synergistic role; the PEDV mutant S protein can synergistically promote viral colonization in the intestine with ORF3.

[0046] According to the research results: CHM2013-SP BJ -ORF3 CHM Loss of pathogenicity to piglets, CHM2013-SP BJ -M CHMBoth strains exhibited reduced pathogenicity and could colonize the intestines. Further evaluation of the immunoprotective effect of these two attenuated strains revealed a decrease in daily viral load shed 8 days after immunization in the immunized group, and no viral shedding was detected by RT-PCR 12 days after immunization, indicating the safety of the chimeric attenuated strains. Specific neutralizing antibodies were detected in the serum of immunized piglets, with titers reaching up to 1:84, indicating that the chimeric attenuated strains could induce an immune response in piglets. Compared to the control group, the immunized group showed reduced diarrhea symptoms and intestinal pathological changes, as well as decreased daily viral load and intestinal viral load. Combined with pathogenicity analysis, the chimeric attenuated strain CHM2013-SP... BJ -ORF3 CHM It is safe, can induce protective immunity in piglets, and can serve as a potential candidate strain for attenuated live vaccine. I. PEDV chimeric virus CHM2013-SP BJ CHM2013-S BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM CHM2013-SP BJ -N CHM And CHM2013-(S+ORF3) BJ The construction and rescue process includes the following steps:

[0047] 1. Prepare materials

[0048] (1) Cells, plasmids, and antibodies. Vero CCL81 cells were preserved in our laboratory. Infectious cloning plasmids: pCHM2013 and pBJ2011C were both constructed and preserved in our laboratory. In the early stage, the laboratory designed primers based on the whole genome sequences of the attenuated strain CHM2013 and the virulent strain BJ2011C, and performed RT-PCR amplification in segments. By introducing restriction sites through synonymous mutation, the full-length cDNA of CHM2013 and BJ2011C was ligated into the low-copy plasmid pBeloBAC11. At the same time, elements such as the cytomegalovirus promoter (CMV), the hepatitis D ribozyme sequence (HDV), and the bovine growth hormone (BGH) sequence were inserted to obtain the full-length cDNA cloning plasmids of CHM2013 and BJ2011C (pCHM2013 and pBJ2011C). After transfection of Vero CCL81 cells, the chimeric viruses CHM2013 and BJ2011C were successfully rescued. PEDV N protein monoclonal antibody-treated mouse ascites was prepared and preserved in our laboratory. FITC-labeled goat anti-mouse IgG antibody (115-095-003) was purchased from Jackson ImmunoResearch Laboratories.

[0049] (2) Molecular biology reagents. High-fidelity polymerase KOD FX NEO (KFX-201) was purchased from Toyobo Corporation, Japan. Gel extraction kit (D2500-02) was purchased from Omega Bio-tek, USA. Restriction endonucleases were purchased from NEB Corporation, USA. Nucleic acid purification kits... SV Gel and PCR Clean-Up System (A9282) was purchased from Promega, USA. Attractene transfection Reagent (301005) was purchased from QIAGEN, Germany. ClonExpress Multis One Step Cloning Kit (C113-02) was purchased from Nanjing Novizan Biotechnology Co., Ltd. Endotoxin-free plasmid extraction kit. The Endo-Free PlasmidMaxi Kit (D6926-03) was purchased from Omega Bio-tek, USA. MagZol TM Reagent (R4801-02) was purchased from Guangzhou Meiji Biotechnology Co., Ltd. Enzyme-free sterile water was purchased from Beijing Solarbio Science & Technology Co., Ltd. 2×Vazyme LAmpMaster Mix (Dye Plus) (P312-03) was purchased from Nanjing Novizan Biotechnology Co., Ltd. Gelred nucleic acid dye EB substitute (MF079-M5) was purchased from Polymer Biotechnology Co., Ltd. Trans 2K DNA Marker (BM101-01) and Trans2K Plus DNA Marker (BM111-01) were purchased from Beijing TransGen Biotech Co., Ltd. The cDNA first-strand synthesis kit FastKing RT Kit (With gDNase) (KR116) was purchased from Tiangen Biotech (Beijing) Co., Ltd. All other analytical grade reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0050] (3) Reagents for agarose gel electrophoresis. Preparation of 50×TAE buffer: Weigh 242g Tris and 18.612g EDTA, dissolve in 800mL distilled water, stir thoroughly to dissolve, add 57.1mL glacial acetic acid, and bring the volume to 1L. Store at room temperature for later use. When using, dilute to prepare 1×TAE electrophoresis buffer. Agarose gel: Weigh agarose powder according to the mass-volume ratio and dissolve in 1×TAE buffer. Heat in a microwave oven until completely dissolved. Add EB substitute at a ratio of 1:10000, mix well, pour into a gel plate with combs inserted, and use after complete solidification.

[0051] (4) Reagents for bacterial culture. Chloramphenicol: Weigh 10g of chloramphenicol, dissolve it in 400mL of anhydrous ethanol, filter it through a 0.22μm filter to sterilize it, and store it in a refrigerator at -20℃ for later use.

[0052] Chloramphenicol-resistant LB liquid medium (1L):

[0053] NaCl 10g

[0054] Tryptone 10g

[0055] Yeast Extract 5g

[0056] Weigh the above reagents and dissolve them in 900 mL of distilled water, then bring the volume to 1 L. Autoclave and store at 4°C. When using, add chloramphenicol at a dilution ratio of 1:2000.

[0057] Chloramphenicol-resistant tryptone soybean agar solid medium (100 mL): Weigh 20 g Tryptic Soy Agar and 5 g Yeast Extract, dissolve in 90 mL distilled water, bring the volume to 100 mL, autoclave for 15 min, cool to 60 °C, and add chloramphenicol. Pour into plates, cool and solidify, and invert in a 4 °C refrigerator for later use.

[0058] (5) Reagents for cell culture. Bismuth subsalicylate (penicillin and streptomycin) was purchased from Beyotime Biotechnology Co., Ltd. 4000 kU of penicillin and 4 g of streptomycin were dissolved in 40 mL of ultrapure water, filtered through a 0.22 μm filter, and stored at -20°C. DMEM powder, fetal bovine serum (FBS) trypsin (1:250) (27250-018), and EDTA-free trypsin (Trypsin (0.25%), phenolred) (150500065) were purchased from Gibco, USA.

[0059] DMEM medium (1L):

[0060] 1 packet of DMEM powder

[0061] NaHCO3 3.75g

[0062] HEPES 5g

[0063] Add the above reagents to 900 mL of ultrapure water, wait for complete dissolution, and then make up to 1 L. Filter using a 0.22 μm filter and store at 4 °C.

[0064] DMEM medium containing 10% FBS: After filtering and sterilizing DMEM, add FBS to make the final content 10%, and add double antibiotics at a ratio of 1:1000.

[0065] PBS buffer (1L):

[0066]

[0067] Weigh the above reagents, dissolve them in 900mL of ultrapure water, and after they are completely dissolved, bring the volume up to 1L. Autoclave and store at 4℃ for later use.

[0068] Na2EDTA-trypsin solution (0.25%) (1L):

[0069] Na2EDTA 0.2g

[0070] Pancreatic enzyme (1:250) 2.5g

[0071] Weigh the above reagents and dissolve them completely in 1L of autoclaved PBS buffer at 4°C. Filter the solution through a 0.22μm filter to remove bacteria and store at 4°C for later use.

[0072] DMEM medium containing 10 μg / mL trypsin: antibiotics and EDTA-free trypsin were added to filtered sterilized DMEM at ratios of 1:1000 and 1:250, respectively.

[0073] (6) Major Instruments and Equipment. The 2720 Thermal Cycler PCR instrument was purchased from Applied Biosystek, USA. The DYY B-type electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory. Gel Doc TM The XR+ gel imaging system was purchased from Bio-Rad, USA. NanoDrop TM The Lite spectrophotometer was purchased from Thermo Fisher Scientific, USA. The SW-CJ-1FD clean bench was purchased from Suzhou Cleanroom Equipment Co., Ltd. The Heracell CO2 cell incubator was purchased from Thermo Fisher Scientific, USA. The SterilGARD III Advance biosafety cabinet (Type A2) was purchased from Baker Scientific, USA. The Thermo Scientific Forma 900 Series ultra-low temperature (-80℃) freezer was purchased from Thermo Fisher Scientific, USA. The BCD-328WDGF freezer was purchased from Qingdao Haier Co., Ltd. The Nikon A1 laser confocal microscope was purchased from Nikon Corporation, Japan.

[0074] 2. Chimeric full-length infectious clone pCHM2013-SP BJ and pCHM2013-S BJ The construction of [the system] specifically includes the following steps:

[0075] (1) Intermediate carrier pCHM2013-SU BJ Construction

[0076] Primers WD0032F / WD0032R were designed for PCR amplification using pCHM2013 as a template, and the amplified fragment was named WD0032. Primers WD0033F / WD0033R were designed for PCR amplification using pCHM22013 as a template, and the amplified fragment was named WD0033. Primers WD0034F / WD0034R were designed for PCR amplification using pBJ2011C as a template, and the amplified fragment was named WD0034.

[0077] The PCR amplification reaction system is as follows:

[0078]

[0079] Reaction conditions:

[0080]

[0081] Purification of the target fragment: All PCR products were electrophoresed on a 0.8% agarose gel, and the gel was then recovered. Following the Omega gel recovery kit instructions, the gel block was chopped and placed in an EP tube. An equal volume of Binding Buffer was added, and the mixture was incubated in a 56°C water bath until completely melted. After cooling to room temperature, the liquid was transferred to a binding column, allowed to stand at room temperature for 2 min, centrifuged at 12000 rpm for 1 min, and the liquid was discarded. Then, 300 μL of Binding Buffer was added, centrifuged at 12000 rpm for 1 min, and the liquid was discarded. Next, 700 μL of SPW buffer was added, centrifuged at 12000 rpm for 1 min, and the liquid was discarded. This step was repeated once. The product was centrifuged at 12000 rpm for 2 min, air-dried at room temperature, dissolved in 30 μL of ddH2O, and allowed to stand at room temperature for 2 min. The concentration of the recovered gel product was measured and stored at -20°C for later use.

[0082] Plasmid digestion: 10 μg of the PEDV infectious clone plasmid pCHM2013 was subjected to double digestion. The digestion system is as follows:

[0083]

[0084] After reacting the prepared system at 25°C for 4 hours, the temperature was adjusted to 37°C and the reaction continued for another 4 hours.

[0085] Plasmid purification: Purify the mixture after enzyme digestion according to the nucleic acid purification kit. The SV Geland PCR Clean-Up System instructions are as follows: Mix an equal volume of membrane binding solution with the mixture to be purified, add to the nucleic acid binding column, incubate at room temperature for 1 min, centrifuge at 16000×g for 1 min, and discard the waste liquid in the collection tube; add 700 μL of washing buffer, centrifuge at 16000×g for 1 min, and discard the waste liquid in the collection tube; add 500 μL of washing buffer and wash once more, centrifuge at 16000×g for 2 min; after the column is completely dried, add 50 μL of nuclease-free water, incubate at room temperature for 1 min, and centrifuge at 16000×g for 1 min; determine the concentration and purity, and store in a -20℃ refrigerator.

[0086] Homologous recombination: The target fragments WD0032, WD0033, WD0034, and linearized pCHM2013 were used for homologous recombination using the ClonExpress Multis One Step Cloning Kit (C113-02). The volume of the reaction solution to be added was calculated based on the target fragment and enzyme digestion plasmid concentrations to obtain the ligation product. The reaction system is as follows:

[0087]

[0088]

[0089] Transformation: Add all ligation products to 100 μL of Trans10 competent cells thawed on ice, mix gently, incubate on ice for 25-30 min, heat shock at 42°C for 45 s, incubate on ice for 2 min, add 500 μL of antibiotic-free LB, incubate at 200 rpm at 37°C for 45 min, centrifuge at 4000 rpm for 5 min, discard the supernatant and keep about 100 μL of supernatant, resuspend and mix the cells, spread on chlorine-resistant tryptone soy agar solid medium, and incubate upside down in a 37°C bacterial incubator.

[0090] Table 1 Primers for constructing infectious cloning plasmids

[0091]

[0092]

[0093] Positive clone identification: Single colonies were selected and incubated in chloramphenicol-resistant LB liquid medium at 160 rpm for 4 h at 37°C. 1 μL of the bacterial culture was then used for colony PCR identification. The PCR products of positive colonies were sequenced; primers are shown in Table 2. If the sequencing results were correct, the culture was inoculated at a 1:1000 ratio into 300 mL of chloramphenicol-resistant LB liquid medium and incubated at 160 rpm for 16 h at 30°C before plasmid extraction.

[0094] Plasmid extraction: Refer to According to the Endo-Free Plasmid Maxi Kit instructions, centrifuge 300 mL of bacterial culture to collect the bacterial cells at 6000 rpm for 5 min, and discard the supernatant; add 15 mL Solution I and thoroughly resuspend and mix the bacterial cells; add 15 mL Solution II, mix well, and let stand at room temperature for 3-5 min; add 8 mL buffer N3, mix gently, let stand at room temperature for 2 min, and centrifuge at 8000 rpm for 10 min. Filter all the supernatant after centrifugation and transfer it to a 50 mL centrifuge tube. Add 0.1 volume of ETR solution, mix gently, and incubate on ice for 10 min, inverting the tube several times during this period. Incubate at 42°C for 5 min, centrifuge at 5000 rpm for 5 min, and then transfer the supernatant to a new 50 mL centrifuge tube. Add 0.5 volume of anhydrous ethanol, mix gently, and incubate at room temperature for 1-2 min. Place the column on the vacuum pump, add 3 mL of GPS buffer, incubate at room temperature for 4 min, and then start the pump for filtration. After all the GPS buffer has been filtered, transfer all the supernatant from the centrifuge tube to the column for filtration. After all the supernatant has been filtered, add 10 mL of HBC buffer and filter. After all the HBC buffer has been filtered, add 15 mL of DNAWash Buffer and filter. Repeat the above steps and continue filtration for 10 min. Dry the filter membrane. Place the column in a new 50 mL centrifuge tube, add 3 mL of nuclease-free water, let stand for 3 min, centrifuge at 10000 r / min for 5 min; purify the plasmid, measure its concentration, and store it in a -20℃ refrigerator.

[0095] Table 2 Primers for PEDV full-length genome sequencing

[0096]

[0097]

[0098] (2) Chimeric full-length infectious clone pCHM2013-SP BJ and pCHM2013-S BJ Construction

[0099] Primers WD0061F / WD0061R were designed and used as templates for PCR amplification with pBJ2011C. The amplified fragment was named WD0061. Primers WD0062F / WD0062R were designed and used as templates for PCR amplification with pCHM2013. The amplified fragment was named WD0062. Following the method in (1), pCHM2013-SU BJDouble digestion with ApaⅠ and BstBI was performed, and homologous recombination was used to combine the cloned gene fragments WD0063 and WD0064 with the enzyme-digested linearized pCHM2013-SU. BJ The vector was ligated. The ligation product was transformed into Trans10 competent cells, and single colonies were picked for PCR identification and sequencing analysis. Primers are shown in Table 2. Positive plasmids were extracted and purified, and named pCHM2013-S. BJ Store in a -20℃ refrigerator for later use.

[0100] Primers WD0067F / WD0067R were designed and used as templates for PCR amplification with pBJ2011C. The amplified fragment was named WD0067. Primers WD0068F / WD0068R were designed and used as templates for PCR amplification with pCHM2013. The amplified fragment was named WD0068. Following the method in (1), pCHM2013-SU BJ Double digestion with ApaⅠ and XhoⅠ was performed, and homologous recombination was used to combine the cloned gene fragments WD0067 and WD0068 with the enzyme-digested linearized pCHM2013-SU. BJ The vector was ligated. The ligation product was transformed into Trans10 competent cells, and single colonies were picked for PCR identification and sequencing analysis. Primers are shown in Table 2. Positive plasmids were extracted and purified, and named pCHM2013-SP. BJ Store in a -20℃ refrigerator for later use.

[0101] 3. Chimeric full-length infectious clone pCHM2013-SP BJ -ORF3 CHM pCHM2013-SP BJ -E CHM pCHM2013-SP BJ -M CHM pCHM2013-SP BJ -N CHM and pCHM2013-(S+ORF3) BJ The construction of [the system] specifically includes the following steps:

[0102] (1) PEDV infectious cloning plasmid pCHM2013-SP BJ and pCHM2013-S BJ Enzyme digestion and purification

[0103] Plasmid digestion: Take 10 μg of PEDV infectious clone plasmid pCHM2013-SP BJ and pCHM2013-S BJDouble enzyme digestion was performed. The prepared system was reacted at 25℃ for 4 hours, then the temperature was increased to 65℃ and the reaction was continued for another 4 hours. The digested plasmid was then purified.

[0104] (2) Amplification and purification of the target fragment

[0105] pCHM2013-SP BJ -E CHM Amplification of the target fragment: Using the pBJ2011C infectious clone plasmid as a template, the S and ORF3 genes were amplified using primers WD0117F / R, and the fragment was named WD0117; using the pCHM2013 infectious clone plasmid as a template, the E gene was amplified using primers WD0118F / R, and the fragment was named WD0118; using the pBJ2011C infectious clone plasmid as a template, the M gene was amplified to the BstBI restriction site using primers WD0117F / R, and the fragment was named WD0119. The target fragment was purified, the concentration of the gel recovery product was determined, and it was stored at -20℃ for later use.

[0106] pCHM2013-SP BJ -M CHM Amplification of the target fragment: Using the pBJ2011C infectious clone plasmid as a template, the S, ORF3, and E genes were amplified using primers WD0120F / R, and the fragment was named WD0120; using the pCHM2013 infectious clone plasmid as a template, the M gene was amplified using primers WD0121F / R, and the fragment was named WD0121; using the pBJ2011C infectious clone plasmid as a template, the N gene was amplified to the BstBI restriction site using primers WD0122F / R, and the fragment was named WD0122. The target fragment was purified, the concentration of the gel recovery product was measured, and it was stored at -20℃ for later use.

[0107] pCHM2013-SP BJ -N CHM Amplification of the target fragment: Using the infectious clone plasmid pBJ2011C as a template, the S, ORF3, E, and M genes were amplified using primers WD0123F / R, and the fragment was named WD0123; using the infectious clone plasmid pCHM2013 as a template, the N gene was amplified to the BstBI restriction site using primers WD0124F / R, and the fragment was named WD0124. The target fragment was purified, the concentration of the gel recovery product was determined, and it was stored at -20℃ for later use.

[0108] pCHM2013-SP BJ -ORF3 CHM Target fragment amplification: using pCHM2013-S BJUsing the infectious clone plasmid as a template, the S and ORF3 genes were amplified using primers WD0125F / R, and the fragment was named WD0125. Using the pBJ2011C infectious clone plasmid as a template, the E gene was amplified to the BstBI restriction site using primers WD0126F / R, and the fragment was named WD0126. The target fragment was purified, the concentration of the gel recovery product was determined, and it was stored at -20°C for later use.

[0109] pCHM2013-(S+ORF3) BJ Amplification of the target fragment: Using the pBJ2011C infectious clone plasmid as a template, the S and ORF3 genes were amplified using primers WD0059F / R (Table 1), and the fragment was named WD0059; using the pCHM2013 infectious clone plasmid as a template, the E gene was amplified to the BstBI restriction site using primers WD0060F / R, and the fragment was named WD0060. The target fragment was purified, the concentration of the gel recovery product was determined, and it was stored at -20℃ for later use.

[0110] (3) Chimeric full-length infectious clone pCHM2013-SP BJ -ORF3 CHM pCHM2013-SP BJ -E CHM pCHM2013-SP BJ -M CHM pCHM2013-SP BJ -N CHM and pCHM2013-(S+ORF3) BJ Construction

[0111] Homologous recombination was used to combine the purified target fragment with the enzyme-digested linearized pCHM2013-SP. BJ Vector or pCHM2013-S BJ The vector was ligated, and the ligation product was transformed into Trans10 competent cells. Single colonies were picked for PCR identification and sequencing analysis. Primers are shown in Table 2. Positive plasmids were extracted and purified by column chromatography. The plasmids were named pCHM2013-SP. BJ -ORF3 CHM pCHM2013-SP BJ -E CHM pCHM2013-SP BJ -M CHM pCHM2013-SP BJ -N CHM and pCHM2013-(S+ORF3) BJ Store in a -20℃ refrigerator for later use.

[0112] 4. PEDV chimeric virus CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM CHM2013-SP BJ -N CHM The salvation of [them] includes the following steps:

[0113] (1) Cell culture and passage

[0114] Frozen Vero CCL81 cells were removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. After complete thawing, the cells were centrifuged at 1000 rpm for 8 minutes, and the supernatant was discarded. The cell pellet was resuspended in 5 mL of DMEM containing 20% ​​FBS and seeded into T25 cell culture flasks. The flasks were then incubated in a cell culture incubator (37°C, 5% CO2) for 6 hours. The culture medium was then replaced with DMEM containing 10% FBS. Once the cells reached confluence, the culture medium was discarded, the cells were washed twice with PBS, and 2 mL of 0.25% trypsin solution was added. The cells were then digested at 37°C for 1-2 minutes, the trypsin was discarded, and the digestion was terminated with DMEM containing 10% FBS. The cells were dispersed by pipetting and passaged at an appropriate density into cell culture flasks and incubated in a cell culture incubator (37°C, 5% CO2) for 36-48 hours.

[0115] (2) Rescue of chimeric viruses

[0116] When the Vero CCL81 cell monolayer in the six-well plate reaches approximately 60% confluence, replace the medium with fresh DMEM containing 10% FBS. Add 2.5 μg of the PEDV chimeric full-length infectious clone plasmid to a 1.5 mL EP tube containing opti-MEM to a final volume of 100 μL, and mix thoroughly using a pipette. Add 2 μL of Attractene Transfection Reagent to the mixture, carefully mix, and incubate at room temperature for 10 min. Add the mixture dropwise to the cell culture wells, return the plate to the incubator, and continue culturing for 24 h. Replace the medium with DMEM containing 10 μg / mL trypsin. Return the cells to the incubator (37℃, 5% CO2) and continue culturing, observing cytopathic effects daily.

[0117] like Figure 2The results showed that syncytial lesions could be observed 60-72 hours after transfection, at a magnification of 100×. IFA identification revealed syncytial-like green fluorescence, and the cell nuclei showed blue staining with DAPI at a magnification of 200×. These results indicate successful rescue of the PEDV chimeric virus, which was named CHM2013-SP. BJ CHM2013-S BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM CHM2013-SP BJ -N CHM And CHM2013-(S+ORF3) BJ .

[0118] (3) Culture of chimeric viruses

[0119] Prepare a monolayer of Vero CCL81 cells 9 2 Cell culture dishes were prepared by washing cells twice with PBS. 200 μL of chimeric virus solution was inoculated into cells, and 1.8 mL of DMEM was added to cover the cells into a monolayer. The dishes were then incubated in a cell culture incubator (37℃, 5% CO2) for 1 hour, shaking the dish every 20 minutes to ensure even coverage. The inoculation solution was discarded, and the cells were washed twice with PBS. 10 mL of DMEM containing 10 μg / mL trypsin was added, and the dishes were incubated in a cell culture incubator (37℃, 5% CO2). The virus was harvested when the cells showed complete cytopathic effects and some cells began to detach. The virus was subjected to three freeze-thaw cycles at -80℃, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected, aliquoted, and stored at -80℃.

[0120] (4) Determination of chimeric virus titer

[0121] Using the determination of the half-maximal tissue culture infection dose (TCID) 50 The viral titer was determined by the following method: 50 μL of viral solution was added to 450 μL of DMEM medium containing 10 μg / mL trypsin and serially diluted 10-fold to a final concentration of 10 μg / mL. -6Vero CCL81 cells monolayered in 96-well plates were inoculated with the virus. The cell culture supernatant was discarded, and the cells were carefully washed twice with 200 μL / well of PBS. Serially diluted virus solution was seeded at 100 μL / well, with each dilution replicated in four wells. The cell culture plates were incubated in a cell culture incubator (37°C, 5% CO2). After 48–72 h of culture, the infection status of each well was determined using indirect immunofluorescence (IFA). The viral titer was calculated using the Reed-Muench method.

[0122] Indirect immunofluorescence (IFA): 48-72 h after cell inoculation with the virus, discard the supernatant and add 100 μL / well of pre-chilled anhydrous ethanol to each well to fix the cells. Incubate at room temperature for 25-30 min. Discard the anhydrous ethanol as completely as possible. Add 100 μL / well of PEDV N monoclonal antibody diluted 1:1000 to each well and incubate at 37°C for 1 h or 4°C overnight. Wash three times with 200 μL / well of PBS. The following steps must be performed under light-protected conditions: Add 100 μL / well of FITC-labeled goat anti-mouse secondary antibody diluted 1:200 to each well and incubate at 37°C for 1 h. Wash three times with PBS. Observe and record using a Nikon inverted fluorescence microscope.

[0123] 5. PEDV chimeric virus CHM2013-SP BJ CHM2013-S BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM and CHM2013-SP BJ -N CHM Identification

[0124] Sequencing and identification: The viral genome fragments were amplified in segments and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The amplification primers were PEDV-20F / PEDV-20R, PEDV-21F / PEDV-21R, PEDV-22F / PEDV-22R, PEDV-23F / PEDV-23R, PEDV-24F / PEDV-24R, PEDV-25F / PEDV-25R, and PEDV-26F / PEDV-2R (see Table 2).

[0125] Viral RNA extraction: Take 250 μL of sample and add 750 μL of MagZol. TMReagent, mix well, and incubate at room temperature for 3-5 min. Add 250 μL of chloroform, vortex vigorously by hand for 15 s, and incubate at room temperature for 5 min. Centrifuge at 12000 rpm for 15 min at 4 °C. Transfer 500 μL of supernatant to a new RNase-free 1.5 mL centrifuge tube, add 500 μL of isopropanol, invert or vortex to mix, and incubate at room temperature for 10 min or overnight at 4 °C to precipitate. Centrifuge at 12000 rpm for 20 min at 4 °C, and discard the supernatant. Add 1 mL of 75% ethanol to wash, and invert to mix well. Centrifuge at 12000 rpm for 5 min at 4 °C, repeat the washing once, discard the supernatant, place the centrifuge tube on clean absorbent paper to remove any residual liquid, and air dry for 10-15 min. Add an appropriate volume of nuclease-free water to dissolve the RNA precipitate, incubate on ice for 10-30 min, and store at -80 °C.

[0126] RT-PCR: The reaction was performed according to the FastKing RT Kit (With gDNase) instructions. First, gDNA was removed by adding 2 μL of 5×gDNA buffer and 8 μL of Total RNA to a 1.5 mL EP tube without RNase. The mixture was thoroughly mixed and briefly centrifuged. The tube was then incubated at 42°C for 3 min, followed by incubation on ice. The reverse transcription reaction mixture was prepared as follows:

[0127]

[0128]

[0129] Add 10 μL of the prepared mix to each reaction solution in the gDNA removal step, mix well, and briefly centrifuge. Incubate at 42°C for 15 min. After incubating at 95°C for 3 min, place on ice to obtain cDNA, which can be used for PCR amplification or stored at low temperature. PCR amplification reaction system (20 μL):

[0130]

[0131] Reaction conditions:

[0132]

[0133] Take 10 μL of the above PCR product for agarose gel electrophoresis identification.

[0134] II. PEDV chimeric virus CHM2013-SP BJ CHM2013-S BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHMCHM2013-SP BJ -M CHM CHM2013-SP BJ -N CHM And CHM2013-(S+ORF3) BJ In vitro proliferation dynamics assay

[0135] Prepare 24-well plates monolayered with Vero CCL81 cells and wash carefully three times with 500 μL / well PBS. Inoculate each well with virus at an MOI of 0.01 according to the viral titer of each chimeric virus, and bring the volume up to 150 μL / well with DMEM. Incubate at 37°C, 5% CO2 for 1 h, shaking every 20 min. Discard the inoculum, wash twice with PBS, add 10 mL of DMEM containing 10 μg / mL trypsin, and incubate at 37°C, 5% CO2. Collect viral loads at 0 h, 12 h, 24 h, 36 h, 48 h, and 60 h post-infection, with three replicates at each time point, and analyze using TCID45. 50 The viral titer at each time point was determined, and the results were used to plot the multi-step growth curve of the chimeric virus using GraphPad 8.4 software.

[0136] like Figure 3 As shown, the in vitro proliferation characteristics analysis results indicate that the chimeric virus CHM2013-SP BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM CHM2013-SP BJ -N CHM CHM2013-S BJ And CHM2013-(S+ORF3) BJ All patients reached peak viral titer 36 hours after infection, and then gradually decreased, with peak viral titer reaching 10 in all cases. 5 TCID 50 / mL or higher. CHM2013-SP BJ The viral titer was higher than CHM2013-S before and at the peak viral titer. BJ CHM2013-SP BJ -ORF3 CHM With CHM2013-SP BJ In comparison, there was no difference in proliferation dynamics; CHM2013-SP BJ -E CHMAfter reaching its peak, the viral load was lower than that of CHM2013-SP. BJ CHM2013-SP BJ -M CHM The price of the drug is lower than that of CHM2013-SP BJ CHM2013-SP BJ -N CHM The viral titer peaked below that of CHM2013-SP. BJ CHM2013-(S+ORF3) BJ With CHM2013-S BJ The in vitro proliferation dynamics were similar. These results indicate that the chimeric virus with the CHM2013 backbone grew well on VeroCCL81 cells, and that structural proteins E, ORF3, M, and N did not affect the in vitro proliferation trend. The structural proteins E, M, and N of the attenuated CHM2013 reduced viral titer to varying degrees. Specifically, protein M affected viral titer throughout the entire in vitro proliferation process of PEDV, protein E affected viral titer in the later stages of PEDV in vitro proliferation, protein N affected the peak viral titer, and ORF3 had no effect on viral titer. Figure 3 In the text, * indicates CHM2013-SP BJ -M CHM With CHM2013-SP BJ The viral titers differed significantly (**, P < 0.01; ***, P < 0.001). # indicates CHM2013-SP BJ With CHM2013-S BJ Viral droplet α represents CHM2013-SP BJ -N CHM With CHM2013-SP BJ Significant differences were found in viral titers (αα, P < 0.01; αα, P < 0.001). Significant differences were also found in viral titers (##, P < 0.01; ###, P < 0.001). β represents CHM2013-SP. BJ -E CHM With CHM2013-SP BJ The viral titer difference was significant (βββ, P<0.001).

[0137] III. Pathogenicity Tests of Chimeric Viruses

[0138] 1. Prepare materials

[0139] (1) PEDV chimeric virus: CHM2013-SP BJ CHM2013-S BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SPBJ -E CHM CHM2013-SP BJ -M CHM CHM2013-SP BJ -N CHM And CHM2013-(S+ORF3) BJ .

[0140] (2) Molecular biology reagents: 2×Vazyme LAmp Master Mix (Dye Plus) (P312-03) was purchased from Nanjing Novizan Biotechnology Co., Ltd., Gelred nucleic acid dye EB substitute (MF079-M5) was purchased from Polymer Biotechnology Co., Ltd., Trans 2K DNA Marker (BM101-01) and Trans 2K Plus DNA Marker (BM111-01) were purchased from Beijing TransGen Biotech Co., Ltd., FastKing one-step reverse transcription-fluorescence quantitative kit (probe method) (FP314) was purchased from Tiangen Biotech (Beijing) Co., Ltd., cDNA first strand synthesis kit FastKing RT Kit (With gDNase) (KR116) was purchased from Tiangen Biotech (Beijing) Co., Ltd., RNAscope Multiplex Fluorescent Reagent Kit v2 Assay (323100) was purchased from Advanced Cell Diagnostics, USA, Poly Mount mounting medium was purchased from Polysciences, USA, and other analytical grade reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0141] (3) Reagents for agarose gel electrophoresis. Preparation of 50×TAE buffer: Weigh 242g Tris and 18.612g EDTA, dissolve in 800mL distilled water, stir thoroughly to dissolve, add 57.1mL glacial acetic acid, and bring the volume to 1L. Store at room temperature for later use. When using, dilute to prepare 1×TAE electrophoresis buffer. Agarose gel: Weigh agarose powder according to the mass-volume ratio and dissolve in 1×TAE buffer. Heat in a microwave oven until completely dissolved. Add EB substitute at a ratio of 1:10000, mix well, pour into a gel plate with combs inserted, and use after complete solidification.

[0142] (4) Major instruments and equipment: The 2720 Thermal Cycler PCR instrument was purchased from Applied Biosys, Inc., USA; the DYYB electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory; and the Gel Doc... TMThe XR+ gel imaging system was purchased from Bio-Rad Laboratories, USA; the SterilGARD III Advance biosafety cabinet (Type A2) was purchased from Baker Laboratories, USA; the CFX96TM Real-time System quantitative PCR instrument was purchased from Bio-Rad Laboratories, USA; the MIO TP-350S intelligent digital display magnetic heating stirrer was purchased from Hangzhou MIO Instruments Co., Ltd.; the HybEZ II Oven hybridization oven was from Advanced Cell Diagnostics, USA; the Thermo Scientific Forma 900 Series ultra-low temperature (-80℃) freezer was purchased from Thermo Fisher Scientific, USA; the BCD-328WDGF freezer was purchased from Qingdao Haier Co., Ltd.; the Bullet Blunder cell disruptor was purchased from Next Advance, USA; the Nikon A1 laser confocal microscope was purchased from Nikon Corporation, Japan; and the Vortex-Genie2 vortex oscillator / vortex mixer was purchased from Scientific Industries, USA.

[0143] 2. Preparation of laboratory animals

[0144] For the loss-of-function test, 28 two-day-old SPF piglets were selected, with 5 piglets in each experimental group and 3 piglets in each control group. For the gain-of-function test, 13 two-day-old SPF piglets were selected, with 5 piglets in each experimental group and 3 piglets in each control group.

[0145] Anal swabs were collected from piglets after birth and tested for common diarrhea viruses in piglets: PEDV, transmissible gastroenteritis virus (TGEV), porcine PDCoV, and rotavirus (RV). All results were negative. Two-day-old piglets were housed in isolators at an ambient temperature of 30°C. They were fed milk every 4 hours daily.

[0146] Diarrhea virus detection: The anal swab was shaken for 10 min, and 1.5 mL of the fecal mixture was transferred to a 2 mL centrifuge tube. The tube was centrifuged at 4℃, 6000 rpm for 5 min, and the supernatant was collected. Virus extraction and RT-PCR were used to detect the presence of diarrhea virus. Primers were designed in our laboratory and synthesized by Beijing Qingke Biotechnology Co., Ltd., and their sequences are shown in Table 3.

[0147] Table 2 Primers for detecting enteroviruses in piglets

[0148]

[0149]

[0150] 3. Piglets undergoing viral infection

[0151] Before challenge, remove the frozen virus solution (loss-of-function test: CHM2013-SP). BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM and CHM2013-SP BJ -N CHM Gain of Function Test: CHM2013-(S+ORF3) BJ and CHM2013-S BJ Dilute the virus to 1×10 using 1×DMEM. 5 TCID 50 / mL. Each piglet in the experimental group was orally administered 5mL of diluted virus solution, with a final challenge dose of 5×10⁹ / mL. 5 TCID 50 The control group received 5 ml of LDM orally per piglet.

[0152] 4. Clinical symptom observation

[0153] Weighing: The weight of the piglets was measured and recorded before the piglets were challenged with the virus, when the piglets died, and at the end of the animal experiment.

[0154] Scoring: Carefully observe each feeding after the virus challenge, and record and score the degree of diarrhea and mental state of the piglets according to the scoring criteria in Table 4.

[0155] Table 3. Scoring criteria for clinical symptoms and diarrhea severity in infected piglets.

[0156]

[0157] The survival curve of piglets after viral challenge is as follows: Figure 4 As shown in A, only CHM2013-SP is available. BJ -N CHM In the first group, piglets died on day 8 post-infection, with a survival rate of 80%. The remaining chimeric virus groups showed no mortality, with a survival rate of 100%. This result indicates that replacing the structural protein of the virulent strain with a weakened virus backbone, despite retaining the weakened N gene, still leads to piglet mortality; therefore, the N gene is not the cause of the increased pathogenicity of the PEDV variant. The average weight gain of piglets was as follows: Figure 4 As shown in B, the results indicate that CHM2013-SP BJ Group and CHM2013-SP BJ -N CHM Group weight loss, CHM2013-SP BJ -ORF3CHM CHM2013-SP BJ -M CHM and CHM2013-SP BJ -E CHM The group gained weight.

[0158] After viral challenge, the daily diarrhea and mental state of the piglets were recorded and scored. Figure 4 As shown in C and D. CHM2013-SP BJ Group and CHM2013-SP BJ -N CHM The group exhibited similar clinical symptoms. CHM2013-SP BJ The group exhibited poor mental state and slow movement on the second day after infection, with symptoms gradually becoming more pronounced. By the seventh day after challenge, symptoms such as loss of appetite, drowsiness, and even a preference for lying down appeared; CHM2013-SP BJ -N CHM The group developed symptoms of poor mental status and slow movement on the 3rd day of infection, and the symptoms gradually worsened thereafter; CHM2013-SP BJ -E CHM Early clinical symptoms and CHM2013-SP BJ Similar to the previous group, symptoms lessened later, but also included loss of appetite and drowsiness; CHM2013-SP BJ -M CHM The group began experiencing slow movement and mild drowsiness on day 5 post-infection; CHM2013-SP BJ -ORF3 CHM The piglets in this group did not show any clinical symptoms of PEDV within 7 days of infection and remained normal. CHM2013-SP BJ Group and CHM2013-SP BJ -N CHM The group experienced severe diarrhea, with watery diarrhea appearing on days 3 and 4 post-infection and persisting until day 7 post-infection; CHM2013-SP BJ -E CHM The piglets in this group experienced severe diarrhea in the early stages, which lessened in the later stages; CHM2013-SPBJ-M CHM The piglets in group CHM2013-SP showed diarrhea symptoms later. In contrast, the piglets in group CHM2013-SP... BJ -ORF3 CHM No diarrhea symptoms were observed in the group. Figure 4 In the text, * indicates CHM2013-SP BJ -ORF3 CHM Infection group and CHM2013-SP BJ Significant differences were observed in diarrhea and mental status among the infected groups (***, P<0.001). # indicates CHM2013-SPBJ -E CHM Infection group and CHM2013-SP BJ Significant differences were observed in diarrhea and mental status among the infected groups (###, P<0.001). α represents CHM2013-SP BJ -M CHM Infection group and CHM2013-SP BJ Significant differences were observed in diarrhea and mental status among the infected groups (ααα, P<0.001). β represents CHM2013-SP BJ -N CHM Infection group and CHM2013-SP BJ Significant differences were found in diarrhea and mental status among the infected groups (ββ, P<0.01; βββ, P<0.001).

[0159] The above results indicate that the chimeric virus CHM2013-SP, which retains the N protein of the attenuated CHM2013 strain, is... BJ -N CHM The diarrhea and clinical symptoms of the piglets did not improve; the chimeric virus CHM2013-SP, which retains the attenuated strain CHM2013 ORF3, E, and M proteins, was tested. BJ -ORF3 CHM CHM2013-SP BJ -N CHM and CHM2013-SP BJ -M CHM Clinical symptoms were alleviated to varying degrees, among which CHM2013-SP BJ -E CHM Group and CHM2013-SP BJ -M CHM Group CHM2013-SP piglets showed diarrhea. BJ -ORF3 CHM The piglets in the group showed almost no diarrhea. In summary, the S protein of the PEDV variant, in conjunction with E, M, and ORF3, promotes viral pathogenicity, with the S protein's synergy with ORF3 being the most crucial factor.

[0160] To further verify whether the S protein and ORF3 can synergistically promote viral pathogenesis, the chimeric virus CHM2013-S was selected for testing. BJ And CHM2013-(S+ORF3) BJ The pathogenicity test in piglets is used as a functional gain test. For example... Figure 5 As shown, where Figure 5 A shows the survival curve of piglets infected with chimeric virus. The survival curve of piglets shows a survival rate of 100%, indicating that CHM2013-S BJ And CHM2013-(S+ORF3)BJ It is not lethal to piglets; Figure 5 B shows the average weight gain of piglets infected with chimeric virus, with the average weight gain results indicating CHM2013-(S+ORF3). BJ The average weight gain of the group was lower than that of CHM2013-S BJ Group; Figure 5 C shows the diarrhea in piglets infected with chimeric virus; the diarrhea results indicate CHM2013-(S+ORF3). BJ The earliest piglets in the group to show diarrhea appeared on the first day of viral challenge, and all piglets excreted liquid feces on the second day, which continued until the 7th day. CHM2013-S BJ The piglets developed soft stools on the 5th day after infection, followed by liquid feces. Figure 5 D shows the clinical symptoms of chimeric virus infection in piglets. The clinical symptom score is consistent with the diarrhea situation. CHM2013-(S+ORF3) BJ The earliest symptoms of loss of appetite and poor mental state appeared in the piglets on the first day after challenge with the virus. On the second day after challenge, some piglets showed symptoms of lying down and anorexia. CHM2013-S BJ The piglets only developed symptoms of loss of appetite and slow movement on the sixth day after being challenged with the virus. Figure 5 In the text, * indicates CHM2013-(S+ORF3). BJ Infection group and CHM2013-S BJ Significant differences were observed in diarrhea and mental status among the infected groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001). These results indicate that, compared to CHM2013-S BJ CHM2013-(S+ORF3) BJ The pathogenicity to piglets is enhanced, with infected piglets exhibiting earlier onset of illness and more severe diarrhea. In conclusion, the PEDV variant BJ2011C's S protein synergistically promotes viral pathogenicity with ORF3.

[0161] 5. Daily detoxification test

[0162] After challenge, anal swabs were collected from piglets three times daily at 7:00, 16:00, and 20:00 using sterile cotton swabs. Viral RNA was extracted and analyzed by RT-PCR. The upstream primer was PEDV-28F: 5'-AGCGGACTCTTACGAGATTACA-3', and the downstream primer was PEDV-28R: 5'-ACACCGTCAGGTCTTCAGTTA-3', with an amplification length of 618 bp.

[0163] like Figure 6 As shown in Figure A, the daily viral shedding results of the loss-of-function pathogenicity test indicate that CHM2013-SP in the challenge group...BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHM and CHM2013-SP BJ -N CHM Detoxification was detected in all groups on day 3, while no detoxification was detected in the MOCK group on day 7. CHM2013-SP BJ -M CHM In the infected group, a positive signal was detected on day 4 post-challenge and persisted until day 7. CHM2013-SP BJ -ORF3 CHM In the infected group, only one piglet continued to shed the virus until day 7, while most piglets showed no detectable viral shedding on day 6 post-infection. The results indicate that CHM2013-SP BJ -ORF3 CHM It is a weakened strain, and piglets can stop shedding the virus relatively early during infection; CHM2013-SP BJ -M CHM The M gene delays viral shedding in piglets and affects the timing of PEDV infection. For example... Figure 6 As shown in B, the daily viral shedding results of the gained-of-function pathogenicity test indicate that CHM2013-S BJ Virus shedding was detectable in the group on the first day of challenge, but only one piglet shed the virus on the second day, and no further shedding was detected thereafter. CHM2013-(S+ORF3) BJ Viral shedding was detectable in the group from the first to the third day after challenge, but no further shedding was detected thereafter. The results indicate that both chimeric viruses have weak ability to multiply and shed viral loads in the intestine.

[0164] In summary, CHM2013-SP BJ Replacing any structural protein of the attenuated strain does not affect the virus's ability to be shed in the intestine, and replacing only ORF3 of the virulent strain does not affect CHM2013-S BJ It enhances the body's ability to reproduce and detoxify in the intestines.

[0165] 6. Observation of intestinal lesions

[0166] Seven days after viral challenge, surviving piglets were euthanized, and necropsies were performed on the deceased piglets. The necropsies of surviving piglets were performed in the following order: control group, attenuated virus group, and then virulent virus group. The focus was on observing lesions in the digestive tract (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum), and photographs were taken of each intestinal segment. 0.5-1 cm segments of the intestine were harvested from the duodenum, jejunum, ileum, cecum, colon, and rectum, rinsed thoroughly with 0.9% NaCl, fixed in 10% neutral formaldehyde, and then stained with hematoxylin and eosin (HE). The magnification was 200×. The remaining intestinal segments were placed in resealable bags, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0167] like Figure 7 As shown, Figure 7 A represents the gross pathological changes in the intestines of piglets infected with chimeric viruses during the loss-of-function experiment. Figure 7 B represents the gross pathological changes in the intestines of piglets infected with chimeric viruses during the gain-of-function experiment. CHM2013-SP BJ With CHM2013-SP BJ -N CHM It causes the most severe damage to the intestines, characterized by thinning of the intestinal wall, dilation and gas in the intestinal lumen, reddening of parts of the intestine, decreased elasticity of the intestinal wall, yellow watery digestive material in the intestines, and severe congestion of the mesentery. CHM2013-SP BJ -M CHM and CHM2013-SP BJ -E CHM The intestinal walls of the piglets were thinned, and the intestinal lumen contained yellowish, watery contents. Slight mesenteric hemorrhage was also observed. CHM2013-SP BJ -ORF3 CHM No intestinal lesions were observed in the piglets of the CHM2013-S and MOCK groups; the intestinal walls were relatively thick, and formed feces were visible in the colon. BJ Infected piglets showed slight thinning of the intestinal wall and several areas of clear yellow fluid in the jejunum; no lesions were observed in other intestinal segments. CHM2013-(S+ORF3) BJ Infected piglets showed multiple lesions in their intestines, including thinning and transparency of the intestinal wall, the presence of yellow fluid, and intestinal lumen dilation. The results indicated that CHM2013-(S+ORF3) was the causative agent. BJ Compared to CHM2013-S BJ It can cause more serious gross pathological changes.

[0168] Intestinal tissue samples were collected and pathological sections were prepared. The tissue lesions were observed using hematoxylin and eosin (HE) staining. Figure 8 As shown. Pathological changes in the intestinal tissue of piglets in the loss-of-function test are as follows. Figure 8 As shown in A. CHM2013-SP BJIn piglets, duodenal epithelial cells sloughed off; the jejunal wall showed edema, shortened villi, thinned intestinal wall, and loose connective tissue; the ileum's lamina propria showed capillary congestion; the cecum, colon, and rectum showed congestion, with increased lymphocytes in the cecum, thinning of the colonic wall muscle layer, and rectal wall edema. CHM2013-SP BJ -N CHM In piglets, the duodenal lamina propria showed increased cell count and congestion, with epithelial cell necrosis and shedding, and mucus secretion within the intestinal lumen; the jejunal wall was thinned, with increased lymphocytes; the ileal lamina propria showed severe congestion; and the cecum showed mild edema and loose connective tissue. CHM2013-SP BJ -E CHM and CHM2013-SP BJ -M CHM In piglets, duodenal epithelial cells were sloughed off; the jejunum showed widespread congestion and edema, with shortened intestinal villi; the ileum was congested; and the large intestine showed loose connective tissue and severe edema. CHM2013-SP BJ -ORF3 CHM The intestinal tract of the group was basically normal, similar to that of the MOCK group, with no thinning of the intestinal wall, shedding of intestinal villi epithelial cells, loose connective tissue edema, or congestion.

[0169] Pathological changes in intestinal tissue of piglets in the gain-of-function test, such as Figure 8 As shown in B, CHM2013-S BJ No abnormalities were observed in the duodenum, cecum, colon, and rectum of the piglets. Localized villus loss was observed in the jejunum, with some villus epithelial loss; the villus length was normal. Mild congestion was observed in the ileum. CHM2013-(S+ORF3) BJ In the MOCK group of piglets, local villi were lost in the duodenum; the villi in the jejunum were shortened, the intestinal wall was thinned, and local epithelial cells of the villi mucosa were sloughed off; the villi in the ileum were shortened and slightly congested; no lesions were found in the rest of the intestine. All segments of the intestine in the MOCK group of piglets appeared normal.

[0170] The above results further assessed the damage of chimeric viruses to the piglet intestine in CHM2013-SP. BJ The chimeric virus CHM2013-SP was obtained by replacing the ORF3 gene of the attenuated CHM2013 virus with that of the chimeric virus. BJ -ORF3 CHM It lost its ability to cause intestinal damage in piglets. The rest are in CHM2013-SP BJ The chimeric virus CHM2013-SP was obtained by replacing the E, M, and N genes of the attenuated CHM2013 virus with those of the chimeric virus. BJ -E CHM CHM2013-SP BJ -M CHM and CHM2013-SP BJ -NCHM It causes varying degrees of damage to the intestines of piglets, primarily affecting the small intestine. CHM2013-(S+ORF3) BJ The damage to the small intestine of piglets in group 2 was more severe than that in CHM2013-S. BJ Group.

[0171] 7. Intestinal colonization analysis

[0172] (1) Enterovirus load assay

[0173] Construction of the pEASY-Blunt-N standard plasmid: Using the BJ2011C infectious clone plasmid as a template, the upstream primer NF (SEQ ID NO.110): 5'-ATGGCTTCTGTCAGTTTTCAG-3', and the downstream primer NR (SEQ ID NO.111): 5'-TTAATTTCCTGTGTCGAAGAT-3', the full-length PEDV N gene (1326bp) was amplified using KOD high-fidelity enzyme. The target fragment was purified and ligated into a vector. Based on the concentration of the gel recovery product, 20ng was added per 1kb, and 1μL of pEASY Blunt Zero Cloning Vector was added. The volume was then brought to 5μL with ddH2O, and the mixture was incubated at 25℃ for 5min before transformation. Positive clones were identified by selecting single colonies and incubating them in LB liquid medium containing ampicillin at 200 rpm for 37℃ for 4h. 1μL of the bacterial culture was then used for colony PCR identification. Identification was performed using universal upstream and downstream primers M13F and M13R from the pEASY Blunt Zero Cloning Vector. Clones whose PCR products were the same size as the target fragment were selected for sequencing. After confirming complete sequence alignment, the clones were amplified and cultured, and plasmids were extracted.

[0174] Plasmid extraction: Following the instructions of the Promega plasmid extraction kit, pour 150 mL of bacterial culture into a centrifuge bottle, centrifuge at 6000 rpm for 5 min, and discard the supernatant; add 5 mL of resuspension buffer and mix thoroughly; add 5 mL of lysis buffer, mix well, and incubate at room temperature for 3-5 min; add 10 mL of neutralization buffer, mix well, and centrifuge at 8000 rpm for 20 min. Transfer the supernatant to a blue column, filter, and discard the blue filter column after the liquid has evaporated. Add 5 mL of endotoxin-free solution to a white filter column, evaporate to dryness, add 20 mL of Wash Buffer, and repeat this step; finally, add 500 μL of nuclease-free water, let stand for 2 min, filter, and measure the concentration of the obtained plasmid.

[0175] Sample preparation: Add steel beads to a 1.5 mL EP tube, weigh and record the weight. In a clean bench, cut off segments of intestine (approximately 0.5 g) and place them in an EP tube containing steel beads, weigh and calculate the intestinal tissue weight. Add 750 μL of DMEM and homogenize in a tissue homogenizer. After complete homogenization, centrifuge at 10000 rpm for 10 min at 4 °C, collect 250 μL of supernatant, and extract total RNA according to the viral RNA extraction method.

[0176] The PEDV genome in intestinal tissue was quantitatively detected using the FastKing one-step reverse transcription-quantitative fluorescence kit (probe method) (FP314). The copy number was calculated based on the concentration of the pEASY-Blunt-N standard plasmid: copies / μL = (plasmid concentration × 6.02 × 10⁻⁶) / μL. 14 ) / (base count × 660). Perform 10-fold serial dilutions using Elution Buffer. 10 -10 1 A standard curve was plotted using plasmids at a concentration of copies / μL as standards. The upstream primer was PEDV-794F (SEQ ID NO.112): 5'-AATCCAGGGCCACTTCGAA-3', the downstream primer was PEDV-864R (SEQ ID NO.113): 5'-TTCGCCCTTGGGAATTCTC-3', and the Taqman probe (SEQ ID NO.114): 5'FAM-AACGTGACCTCAAAGACATCCCAGAGTGG-3'BHQ-1. The reaction system is as follows:

[0177]

[0178] The reaction conditions are as follows:

[0179]

[0180] After the reaction, the amplification curve, CT value, and standard curve were confirmed. The PEDV genome copy number in each intestinal tissue was calculated based on the standard curve. The results were statistically analyzed using GraphPad 8.4 software.

[0181] The number of chimeric virus genome copies per unit weight in each segment of the intestine was detected, and the viral load in each segment of the piglet's intestine infected with chimeric virus was as follows: Figure 9 As shown. Figure 9 As shown in Figure A, in the loss-of-function test, all chimeric viruses were detectable in viral load in the duodenum, jejunum, ileum, cecum, colon, and rectum of piglets. The viral load was lowest in the duodenum, while the viral load in the jejunum, ileum, and large intestine could reach 10. 8 copies / g.

[0182] Results of functional gain test, such as Figure 9 B shows CHM2013-S BJ Viral RNA was not detected in any of the intestines of infected piglets (CHM2013-(S+ORF3)). BJ Viral RNA can be detected in infected piglets, but the highest copy number only reaches 10. 5.5 copies / g. Nucleic acid in situ hybridization results showed CHM2013-(S+ORF3) BJ Viral colonization can be detected in the jejunum and ileum of piglets, while the positive signal is less in the ileum. Figure 9 In B, * indicates CHM2013-(S+ORF3) BJ Infection group and CHM2013-S BJ The enterovirus copy number differed significantly among the infected groups (***, P<0.001). These results indicate that CHM2013-S BJ It cannot colonize the intestines; CHM2013-(S+ORF3) BJ It can colonize the intestines, but its colonization ability is relatively weak.

[0183] (2) In situ hybridization of nucleic acid probes (RNA scope)

[0184] Dewaxing: Place tissue sections in xylene at room temperature for 5 minutes, occasionally moving the slide up and down. Repeat the above steps once using fresh xylene. Immediately after removing the sections from the xylene, place them in anhydrous ethanol and incubate at room temperature with stirring for 2 minutes. Repeat the above steps once using fresh anhydrous ethanol. Remove the sections and dry them in a 60°C oven for 5 minutes, or until completely dry.

[0185] Hydrogen peroxide treatment: Add a few drops of H2O2 to the dewaxed sections to cover the tissue, incubate at room temperature for 10 min, discard the H2O2, wash 3-5 times in ddH2O, and repeat once with fresh ddH2O.

[0186] Target remediation: Place 1×Target Retrieval Reagent in a beaker, cover with aluminum foil, and heat on high for 10-15 minutes. When the temperature reaches 98-102℃, maintain at 99℃ on low. Slowly immerse the slide in the solution, maintaining 99℃ for 35 minutes. Wash 3-5 times with ddH2O, then soak in fresh anhydrous ethanol for 3 minutes. Remove the slide and dry in a 60℃ oven for 5 minutes, until completely dry.

[0187] Protease pretreatment: Draw a hydrophobic zone around the tissue using a hydrophobic pen, dry in a 60°C oven for 5 minutes or at room temperature overnight. Add RNA. Cover the tissue with Protease Plus, place it in a hybridization oven, incubate at 40°C for 30 min, wash the sections 3-5 times in ddH2O, and repeat once with fresh ddH2O.

[0188] Hybridization: Preheat PEDV-N Probe to 40°C in a water bath and cool to room temperature. Add PEDV-N Probe to cover the tissue, place in a hybridization oven, and incubate at 40°C for 2 hours. Wash with 1×Wash Buffer at room temperature for 2 minutes, repeating the wash once to completely remove liquid from the slides. Add RNAscope Multiple FL v2 AMP1 to cover the tissue, place in a hybridization oven, and incubate at 40°C for 30 minutes. Wash with 1×Wash Buffer at room temperature for 2 minutes, repeating the wash once to completely remove liquid from the slides. Add RNAscope Multiple FL v2 AMP2 to cover the tissue, incubate at 40°C for 30 minutes, place in a hybridization oven, and incubate at 40°C for 30 minutes. Wash with 1×Wash Buffer at room temperature for 2 minutes, repeating the wash once to completely remove liquid from the slides. Add RNAscope Multiple FL v2 AMP3 to cover the tissue, place in a hybridization oven, and incubate at 40°C for 30 minutes. Wash with 1×Wash Buffer at room temperature for 2 minutes, repeating the wash once to completely remove liquid from the slides.

[0189] Development: Cover the tissue with RNAscope Multiple FL v2 HRP-C1, incubate in a hybridization oven at 40°C for 30 min, wash with 1×Wash Buffer at room temperature for 2 min, repeat once to completely remove liquid from the slide. All steps require protection from light. Cover the tissue with 1×TSA Plus Cyanine3 diluted with TSA, incubate in a hybridization oven at 40°C for 30 min, wash with 1×Wash Buffer at room temperature for 2 min, repeat once to completely remove liquid from the slide. Cover the tissue with RNAscope Multiple FL v2 HRP blocker, incubate in a hybridization oven at 40°C for 30 min, wash with 1×Wash Buffer at room temperature for 2 min, repeat once to completely remove liquid from the slide. Cover the tissue with DAPI, incubate at room temperature for 3-5 min, wash 5 times in ddH2O, 5 min each time. Add mounting medium, cover with coverslips, air dry overnight at room temperature, and store at 4°C or -20°C protected from light.

[0190] The RNA scope technique, used for in situ hybridization of nucleic acids, was further used to detect viral colonization in the gut, such as... Figure 10As shown, the PEDV N gene, bound by the hybridization probe, appears red through a stepwise signal amplification process. The cell nuclei are stained blue by DAPI. The magnification is 200x. Loss-of-function assay results indicate that in CHM2013-SP... BJ The chimeric virus CHM2013-SP was obtained by replacing the ORF3, E, M, and N genes of the attenuated CHM2013 virus with those of the chimeric virus. BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM and CHM2013-SP BJ -N CHM The tropism of CHM2013-(S+ORF3) to piglet intestinal tissue was not altered. The results of the gain-of-function test indicated that CHM2013-(S+ORF3) BJ Virus colonization, CHM2013-S, can be detected in the jejunum and ileum of piglets. BJ It cannot colonize the intestines.

[0191] In summary, none of the ORF3, E, M, and N genes alters CHM2013-SP. BJ Intestinal colonization ability; ORF3 can synergistically promote viral intestinal colonization with the S gene. Attenuated strain CHM2013-SP BJ -ORF3 CHM and CHM2013-SP BJ -M CHM It can colonize the intestines of piglets, providing favorable conditions for stimulating mucosal immunity. Attenuated strains hold promise as candidate strains for PEDV attenuated vaccines.

[0192] IV. Evaluation of the immunoprotective effect of attenuated live vaccine candidate strains

[0193] CHM2013-SP BJ -M CHM The pathogenicity of CHM2013-SP has decreased. BJ -M CHM The onset time of the virus strain is delayed; CHM2013-SP BJ -ORF3 CHM The strains lost their pathogenicity to 2-day-old piglets and could colonize the intestines, making them promising candidates for vaccines. The immunoprotective effects of these two attenuated strains were evaluated using 2-day-old SPF piglets as an animal model.

[0194] 1. Prepare materials

[0195] (1) PEDV chimeric virus: CHM2013-SP BJ-ORF3 CHM CHM2013-SP BJ -M CHM And BJ2011C.

[0196] (2) Molecular biology reagents: 2×Vazyme LAmp Master Mix (Dye Plus) (P312-03) was purchased from Nanjing Novizan Biotechnology Co., Ltd., Gelred nucleic acid dye EB substitute (MF079-M5) was purchased from Polymer Biotechnology Co., Ltd., Trans 2K DNA Marker (BM101-01) and Trans 2K Plus DNA Marker (BM111-01) were purchased from Beijing TransGen Biotech Co., Ltd., FastKing one-step reverse transcription-fluorescence quantitative kit (probe method) (FP314) was purchased from Tiangen Biotech (Beijing) Co., Ltd., cDNA first strand synthesis kit FastKing RT Kit (With gDNase) (KR116) was purchased from Tiangen Biotech (Beijing) Co., Ltd., RNAscope Multiplex Fluorescent Reagent Kit v2 Assay (323100) was purchased from Advanced Cell Diagnostics, USA, Poly Mount mounting medium was purchased from Polysciences, USA, and other analytical grade reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0197] (3) Reagents for agarose gel electrophoresis. Preparation of 50×TAE buffer: Weigh 242g Tris and 18.612g EDTA, dissolve in 800mL distilled water, stir thoroughly to dissolve, add 57.1mL glacial acetic acid, and bring the volume to 1L. Store at room temperature for later use. When using, dilute to prepare 1×TAE electrophoresis buffer. Agarose gel: Weigh agarose powder according to the mass-volume ratio and dissolve in 1×TAE buffer. Heat in a microwave oven until completely dissolved. Add EB substitute at a ratio of 1:10000, mix well, pour into a gel plate with combs inserted, and use after complete solidification.

[0198] (4) Major instruments and equipment: The 2720 Thermal Cycler PCR instrument was purchased from Applied Biosys, Inc., USA; the DYYB electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory; and the Gel Doc... TMThe XR+ gel imaging system was purchased from Bio-Rad Laboratories, USA; the SterilGARD III Advance biosafety cabinet (Type A2) was purchased from Baker Laboratories, USA; the CFX96TM Real-time System quantitative PCR instrument was purchased from Bio-Rad Laboratories, USA; the MIO TP-350S intelligent digital display magnetic heating stirrer was purchased from Hangzhou MIO Instruments Co., Ltd.; the HybEZ II Oven hybridization oven was from Advanced Cell Diagnostics, USA; the Thermo Scientific Forma 900 Series ultra-low temperature (-80℃) freezer was purchased from Thermo Fisher Scientific, USA; the BCD-328WDGF freezer was purchased from Qingdao Haier Co., Ltd.; the Bullet Blunder cell disruptor was purchased from Next Advance, USA; the Nikon A1 laser confocal microscope was purchased from Nikon Corporation, Japan; and the Vortex-Genie2 vortex oscillator / vortex mixer was purchased from Scientific Industries, USA.

[0199] 2. Preparation of experimental animals

[0200] Twelve 2-day-old SPF piglets, CHM2013-SP, were selected. BJ -ORF3 CHM Group 4 and CHM2013-SP BJ -M CHM Three piglets were placed in the control group and five in the control group. Anal swabs were collected from piglets after birth and tested for common porcine diarrhea viruses; all results were negative.

[0201] 3. Immunization and challenge of piglets

[0202] Before immunization, remove the known TCID. 50 Frozen virus fluid CHM2013-SP BJ -ORF3 CHM and CHM2013-SP BJ -M CHM It was diluted to 1×10 using DMEM. 5 TCID 50 / mL. Each piglet in the immunization group was given 5mL orally, with a final immunization dose of 5 × 10⁹ / mL. 5 TCID 50 In the control group, each piglet was orally administered 5 mL of DMEM culture medium. Twenty-one days after immunization, known TCID values ​​were collected. 50 The frozen BJ2011C virus fluid was diluted to a titer of 1×10 using DMEM. 5 TCID 50 / mL. Each piglet in each group was orally challenged with 5mL of the virus, and the final challenge dose was 5×10⁻⁶. 5 TCID 50 .

[0203] 4. Safety assessment

[0204] Daily detoxification test: After immunization, anal swabs are collected from piglets using sterile cotton swabs, starting from immunization Day 0 to immunization Day 28, for a total of three times a day.

[0205] according to Figure 11 As shown in result A, immunization against CHM2013-SP BJ -ORF3 CHM Piglets in the group reached peak viral load 7-10 days after immunization with CHM2013-SP. BJ -M CHM The group reached its peak detoxification 5-7 days after the viral challenge, with all groups reaching 10. 9 The viral load decreased by copies / ml, and the daily viral copy number decreased starting from day 8. RT-PCR nucleic acid gel electrophoresis results showed (see...). Figure 12 CHM2013-SP BJ -ORF3 CHM Group and CHM2013-SP BJ -M CHM No viral shedding was detected in either group on days 11 and 12 post-immunization. qPCR could detect viral copy number 12 days post-immunization, and 10 copies were detectable within the first three days before challenge (Day-3-Day 0), i.e., days 18 to 21 post-immunization (Day 18-Day 21). 5 The excretion rate was approximately copies / ml. The results indicated that CHM2013-SP... BJ -ORF3 CHM and CHM2013-SP BJ -M CHM As a vaccine candidate strain, it exhibits low or no viral shedding within a short period after immunization, demonstrating its safety.

[0206] 5. Immunogenicity assessment

[0207] (1) Clinical symptom observation

[0208] like Figure 13 As shown in the growth curves of BJ2011C-infected piglets in group A, all piglets in both the immunized and control groups survived. As a highly virulent PEDV strain, BJ2011C exhibits strong pathogenicity and high mortality in 2-day-old piglets, but is not lethal to 21-day-old piglets. According to... Figure 11Analysis of diarrhea results in group B showed that the control group experienced softened stools and liquid stools on days 2-3 after challenge with BJ2011C. Immunization with CHM2013-SP... BJ -M CHM The group also experienced softened stools on days 2-3, while the group immunized with CHM2013-SP... BJ -ORF3 CHM The group experienced softer stools on days 4-5. Figure 13 In the text, * indicates the MOCK group and CHM2013-SP BJ -ORF3 CHM The diarrhea scores showed statistically significant differences (**, P < 0.01; ***, P < 0.001), # indicating the difference between the MOCK group and the CHM2013-SP group. BJ -M CHM The diarrhea scores of the groups showed statistical differences (##, P<0.01; ###, P<0.001). The results indicate that BJ2011C has reduced pathogenicity and no lethality in 21-day-old piglets, and the immunized piglets showed milder diarrhea symptoms compared to the control group.

[0209] (2) Detection of viral shedding copy number and enterovirus copy number

[0210] like Figure 11 As shown in B, starting from the second day after oral challenge, the MOCK group had a higher viral copy number than CHM2013-SP. BJ -ORF3 CHM Group and CHM2013-SP BJ -M CHM Group. CHM2013-SP BJ -ORF3 CHM Group and CHM2013-SP BJ -M CHM The copy number of the virus shed after attacking BJ2011C did not fluctuate and remained consistent with the pre-attack shed count at 10. 5 Approximately copies / ml. RT-PCR nucleic acid gel electrophoresis results show (see...) Figure 12 CHM2013-SP BJ -ORF3 CHM Group and CHM2013-SP BJ -M CHM After attacking BJ2011C, the group showed almost no detoxification, while the MOCK group continued to detoxify. Figure 11 In B, * indicates CHM2013-SP BJ -ORF3 CHMThe nucleic acid load in the group and the MOCK group was statistically different (*, P<0.05; **, P<0.01; ***, P<0.001), # indicates CHM2013-SP BJ -M CHM The nucleic acid load in the immunized group and the MOCK group showed statistically significant differences (#, P < 0.05; ###, P < 0.001). These results indicate that after infection with a virulent virus, the viral copy number shed in the immunized group was lower than that in the control group, and immunization with a live attenuated vaccine can reduce the viral copy number shed.

[0211] Intestinal samples were collected from different segments of piglets during necropsy, and the number of chimeric viral RNA copies per unit weight of intestinal segment was detected using absolute quantitative PCR probe method. Results are as follows: Figure 14 As shown, PEDV genomes could be detected in the duodenum, jejunum, ileum, cecum, colon, and rectum of piglets, and the viral load in each segment of the intestine of the immunized group was less than 10. 5 The viral load was copies / g and there was almost no difference between different segments of the intestine, while in the control group, the viral load in each segment of the intestine was higher than 10. 6 The viral load per copy / g differed from that of the immunized group, with the highest viral load in the ileum, followed by the cecum, colon, and rectum. Figure 14 In the text, * indicates the MOCK group and CHM2013-SP BJ -ORF3 CHM The intestinal nucleic acid load showed statistically significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001), # indicating the difference between the MOCK group and CHM2013-SP. BJ -M CHM The intestinal nucleic acid load of the group and the sample showed statistically significant differences (##, P<0.01). The results indicate that CHM2013-SP BJ -ORF3 CHM and CHM2013-SP BJ -M CHM Immunization of piglets reduces the number of BJ2011C virus copies in the intestine.

[0212] (3) Pathological changes in the intestines of piglets

[0213] After the BJ2011C challenge experiment, infected piglets were euthanized, and their intestines were dissected for gross pathological changes. Intestinal segments were collected, fixed in formaldehyde, and then stained with hematoxylin and eosin (HE) for histopathological examination. Gross intestinal lesions in the infected piglets showed multiple areas of thinning and transparency of the intestinal wall with yellow fluid in the small intestine of the MOCK group (see...). Figure 15 A), CHM2013-SP BJ -M CHM Yellow fluid was found in certain segments of the intestine in group CHM2013-SP BJ-ORF3 CHM No gross intestinal lesions were observed in any of the experimental groups, and no pathological changes were found in the large intestine. Histopathological changes in the intestines of piglets in each group were observed by HE staining. Figure 15 As shown in Figure B, in the MOCK group of piglets, there was localized necrosis and sloughing of the duodenal mucosa epithelium, extensive necrosis and sloughing of the jejunum mucosa epithelium with severe mucosal damage, detachment of intestinal villi into the intestinal lumen and congestion of the lamina propria in the ileum, and no lesions were observed in the cecum, colon, and rectum; CHM2013-SP BJ -M CHM The jejunum villi were shortened, the ileum villi were necrotic and sloughed off, and there was slight edema in the submucosa. The remaining intestinal tissues were neatly arranged and no abnormalities were observed; CHM2013-SP BJ -ORF3 CHM The lamina propria of the jejunum and ileum in the control group showed slight congestion, while the remaining intestinal tissues were neatly arranged and showed no abnormalities. In summary, the gross and histopathological changes in the intestines of piglets immunized with the attenuated live vaccine were less severe than those in the control group, particularly CHM2013-SP. BJ -ORF3 CHM The immunized group showed milder intestinal pathological changes, indicating that the attenuated vaccine candidate strain can provide effective immune protection.

[0214] (4) Serum neutralizing antibody detection

[0215] Serum was collected from each piglet at 14, 21, and 28 days post-immunization, and the level of neutralizing antibodies against PEDV BJ2011C in the serum was measured. The specific procedures are as follows:

[0216] Approximately 5 mL of blood was collected from the anterior vena cava of each piglet. The syringe was tilted and left at room temperature for 2 hours, then placed in a 4°C refrigerator for 4 hours. After the serum had separated relatively completely, it was aliquoted into 1.5 mL EP tubes in a biosafety cabinet, centrifuged at 4500 rpm for 5 minutes at 4°C, and the supernatant was collected and stored at -20°C for later use.

[0217] Serum was serially diluted 2-fold in 1.5 mL EP tubes using DMEM culture medium. -1 Dilute to 2 -11 The BJ2011C virus solution was diluted to 2000 TCID using DMEM culture medium. 50 / mL. Mix equal volumes of serum and diluted virus solution at different dilutions. Simultaneously, mix equal volumes of DMEM with diluted virus solution as a positive control. After vortexing, incubate in a cell culture incubator (37℃, 5% CO2) for 1 h. For a 96-well cell culture plate, seed Vero CCL81 cells monolayers and carefully wash twice with 200 μL / well of PBS, discarding the PBS. Add 100 μL of serum and virus mixture to each well, with four replicates for each serum dilution. Simultaneously, add 100 μL of DMEM and 100 μL of a mixture of DMEM and diluted virus solution as negative and positive controls, respectively. Incubate in a cell culture incubator (37℃, 5% CO2) for 1 h. Carefully wash twice with 200 μL / well of PBS, discarding the PBS. Add 100 μL of DMEM medium containing 10 μg / mL trypsin to each well using a multipipeline and incubate in a cell culture incubator (37℃, 5% CO2) for 48-72 h. The number of positive cell wells was counted according to method 2.2.2.10, and the neutralizing antibody titer of BJ2011C in piglet serum was calculated according to the Reed-Muench method.

[0218] CHM2013-SP BJ -ORF3 CHM The highest neutralizing titers against BJ2011C on Day 14, Day 21, and Day 28 after group immunization were 1:19.03, 1:32, and 1:64, respectively, showing a gradually increasing trend. CHM2013-SP BJ -M CHM The highest neutralizing titers against BJ2011C on Day 14, Day 21, and Day 28 after immunization were 1:43.35, 1:12.702, and 1:84.45, respectively. The neutralizing antibody level in the MOCK group was 0, which differed from the serum neutralizing antibody level in the immunized group. Figure 16 As shown in A, * indicates CHM2013-SP BJ -ORF3 CHM There were statistically significant differences in antibody titers between the CHM2013-SP group and the MOCK group (*, P < 0.05; **; P < 0.01; ***, P < 0.001), where # indicates CHM2013-SP. BJ -M CHM There were statistically significant differences in antibody titers between the group and the MOCK group (#, P < 0.05; ##, P < 0.01; ###, P < 0.001).

[0219] (5) Detection of IgG in serum

[0220] The level of anti-PEDV IgG in serum was detected using indirect immunofluorescence. Experimental principle: BJ2011C virus was used as the antigen, diluted serum as the primary antibody, and FITC-labeled goat anti-pig IgG antibody as the fluorescent secondary antibody. The BJ2011C virus stock solution was diluted 100-fold using DMEM medium containing 10 μg / mL trypsin. 100 μL of the diluted virus solution was added to each well of a 96-well cell culture plate containing a monolayer of Vero CCL81 cells and incubated in a cell culture incubator (37℃, 5% CO2). After 12 hours, cytopathic effects were observed under a microscope. IFA assays were performed after syncytial disease was observed. The supernatant was discarded, and 100 μL / well of pre-chilled anhydrous ethanol was added to each well to fix the cells. The cells were incubated at room temperature for 25-30 minutes, and the anhydrous ethanol was discarded as completely as possible. 2% BSA was added to each well at 100 μL / well, and the cells were incubated at 37℃ for 1 hour. The cells were then washed three times with 200 μL / well of PBS. Serum was diluted in 1.5 mL EP tubes with PBS buffer at dilutions of 1:50, 1:100, 1:200, 1:400, and 1:800. 100 μL of serum dilution was added to each well of a 96-well cell plate, with three replicates for each dilution. The plate was incubated overnight at 4°C. Simultaneously, 100 μL of PEDV N monoclonal antibody diluted 1:1000 and PBS were added to each well as a positive and negative control, respectively. The plates were washed three times with PBS. The following steps were performed under light-protected conditions: 100 μL of FITC-labeled goat anti-pig secondary antibody diluted 1:200 was added to each well, and the plate was incubated at 37°C for 1 hour. The plate was then washed three times with PBS. Fluorescence intensity was observed and recorded using a Nikon inverted fluorescence microscope. The highest fluorescence intensity was considered the optimal dilution, and the highest serum dilution with 50% fluorescence intensity was taken as the IgG titer.

[0221] Figure 15 B represents the IgG level in piglet serum. Using BJ2011C virus as the antigen, diluted serum as the primary antibody, and FITC-labeled goat anti-pig IgG antibody as the fluorescent secondary antibody, indirect immunofluorescence was performed to detect the anti-PEDV IgG level in piglet serum. CHM2013-SP BJ -ORF3 CHM The highest serum anti-PEDV IgG levels reached 1:400, 1:400, and 1:200 on Day 14, Day 21, and Day 28 after group immunization, respectively. CHM2013-SP BJ -M CHMThe highest levels of anti-PEDV IgG in the serum of piglets in the immunized group reached 1:200, 1:400, and 1:400 on Day 14, Day 21, and Day 28 post-immunization, respectively. In the MOCK group, the anti-PEDV IgG level was 0 on Day 14 and Day 21, meaning the serum contained no anti-PEDV IgG. On Day 28 post-immunization, the anti-PEDV IgG level was 1:50. Both before and after challenge, the anti-PEDV IgG level in the serum of immunized piglets was higher than that in the control group. *Indicates CHM2013-SP BJ -ORF3 CHM There were statistically significant differences in antibody titers between the CHM2013-SP group and the MOCK group (*, P < 0.05; **; P < 0.01; ***, P < 0.001), where # indicates CHM2013-SP. BJ -M CHM There were statistically significant differences in antibody titers between the CHM2013-SP group and the MOCK group (#, P < 0.05; ##, P < 0.01; ###, P < 0.001). The results indicate that CHM2013-SP... BJ -ORF3 CHM and CHM2013-SP BJ -M CHM It provides some immune protection for piglets. In summary, the attenuated live vaccine candidate strain CHM2013-SP... BJ -ORF3 CHM It not only has the characteristics of low pathogenicity and good safety, but can also induce piglets to produce neutralizing antibodies and protective immunity.

[0222] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for identifying PEDV chimeric virus attenuation based on reverse genetics, characterized in that, Includes the following steps: S1. First, using CHM2013 as the backbone, construct and rescue the chimeric virus CHM2013-SP, which has replaced the coding region of the BJ2011C structural protein and the S protein, respectively. BJ and CHM2013-S BJ Then CHM2013-SP BJ Using this as a backbone, we constructed and rescued the chimeric virus CHM2013-SP, which had its structural proteins replaced by CHM2013. BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM and CHM2013-SP BJ -N CHM To conduct loss-of-function experiments, we will explore the molecular basis of the synergistic promotion of pathogenicity by the S protein and other structural proteins in the PEDV mutant strain BJ2011C. S2. Using 2-day-old SPF piglets as a model, administer 5×10 ml orally. 5 TCID 50 The chimeric virus CHM2013-SP BJ CHM2013-SP BJ -ORF3 CHM CHM2013-SP BJ -E CHM CHM2013-SP BJ -M CHM and CHM2013-SP BJ -N CHM Loss-of-function experiments were conducted to analyze the pathogenicity of the chimeric virus in piglets based on clinical condition, daily viral shedding, intestinal pathological changes, and intestinal colonization. The results showed that, compared to CHM2013-SP... BJ In comparison, CHM2013-SP BJ -N CHM Pathogenicity has not been reduced; CHM2013-SP BJ -M CHM and CHM2013-SP BJ -E CHM The pathogenicity of CHM2013-SP has decreased; BJ -ORF3 CHM It lost its pathogenicity; the PEDV mutant strain obtained the S gene co-structural proteins ORF3, E and M to promote viral pathogenicity, among which the S gene co-constructing ORF3 is the most critical to promote pathogenicity. S3. Based on the results of the loss-of-function test, using CHM2013-S BJ Using this as a framework, construct and rescue the chimeric virus CHM2013-(S+ORF3) that replaces BJ2011C ORF3. BJ To conduct gain-of-function experiments, further explore the molecular basis of the synergistic promotion of pathogenesis by S protein and ORF3; S4. Using 2-day-old SPF piglets as a model, administer 5×10 ml orally. 5 TCID 50 The chimeric virus CHM2013-S BJ And CHM2013-(S+ORF3) BJ The pathogenicity of the chimeric virus to piglets was analyzed by examining clinical conditions, daily viral shedding, intestinal pathological changes, and intestinal colonization. Further investigation was conducted to determine whether the highly virulent strain BJ2011C S gene and structural protein ORF3 synergistically promote viral pathogenicity. The results showed that CHM2013-(S+ORF3) BJ Its pathogenicity is higher than that of CHM2013-S BJ The PEDV mutant strain BJ2011C was found to have a co-structural protein ORF3 in the S gene that promotes viral pathogenicity. S5. After piglet challenge, record the survival status of piglets daily. Weigh and record the weight of piglets before challenge, at the time of death, and at the end of the animal experiment. Carefully observe and record the degree of diarrhea and mental state of piglets according to the scoring criteria. Compare the survival curve, average weight gain, clinical score, and diarrhea score results. In the loss-of-function test, compare with CHM2013-SP. BJ In comparison, CHM2013-SP BJ -N CHM It is both lethal and pathogenic, CHM2013-SP BJ -M CHM and CHM2013-SP BJ -E CHM The pathogenicity has decreased, and infected piglets show diarrhea symptoms. CHM2013-SP BJ -ORF3 CHM It lost its pathogenicity, and infected piglets showed normal clinical symptoms; in the gain-of-function test, compared with CHM2013-S BJ CHM2013-(S+ORF3) BJ The pathogenicity to piglets is enhanced, and infected piglets show earlier onset of illness and more severe diarrhea symptoms; S6. After challenge, anal swabs were collected from piglets daily, and RT-PCR was used to detect viral shedding in infected piglets; loss-of-function test results showed that CHM2013-SP BJ -ORF3 CHM Infected piglets can end viral shedding earlier, CHM2013-SP BJ -M CHM It delays the time of viral shedding in piglets; the results of the gain-of-function test indicate that the chimeric virus CHM2013-S BJ And CHM2013-(S+ORF3) BJ Its ability to reproduce and detoxify in the intestines is relatively weak; S7. Observe the gross and histopathological changes in piglets, and perform the loss-of-function test (CHM2013-SP). BJ With CHM2013-SP BJ -N CHM It causes the most severe damage to the intestines, CHM2013-SP BJ -M CHM and CHM2013-SP BJ -E CHM Thinning of the intestinal wall in piglets is the second most damaging factor to the intestines, CHM2013-SP BJ -ORF3 CHM No intestinal lesions were observed in the CHM2013-(S+ORF3) group and the MOCK group piglets; in the functional gain test, CHM2013-(S+ORF3) was effective. BJ Compared to CHM2013-S BJ This can cause more serious gross pathological changes; S8. Detection of the PEDV genome in intestinal tissue showed that all chimeric viruses were detectable in the intestine during the loss-of-function assay, while the chimeric virus CHM2013-S was not detected in the gain-of-function assay. BJ It cannot colonize the intestine; it is a chimeric virus CHM2013-(S+ORF3). BJ It can colonize the intestine, and the replacement of any structural protein will not change CHM2013-SP. BJ The ability of PEDV to colonize the gut was assessed, and the structural protein S protein could work in conjunction with ORF3 to promote viral gut colonization. Absolute quantitative PCR and nucleic acid in situ hybridization were used to detect the PEDV genome in intestinal tissue. Intestinal samples were collected from different segments of piglets, and the number of chimeric virus genome copies per unit weight of each intestinal segment was detected using absolute fluorescence quantitative PCR probe method. In situ hybridization of nucleic acid probes was used to further detect the colonization of the virus in the intestine.

2. The method for identifying PEDV chimeric virus attenuation based on reverse genetics as described in claim 1, characterized in that, Steps S1 and S3 also include: Sequencing of all chimeric viruses confirmed that PEDV chimeric viruses had no base mutations. Indirect immunofluorescence identification results showed that syncytial-like green fluorescence could be observed in Vero CCL81 cells, indicating that the chimeric viruses were successfully rescued.

3. The method for identifying PEDV chimeric virus attenuation based on reverse genetics as described in claim 1, characterized in that, Steps S1 and S3 also include: Multi-step growth curves of PEDV chimeric virus in Vero CCL81 cells were determined. Virus was inoculated into 24-well plates containing a monolayer of Vero CCL81 cells at an MOI of 0.

01. Viral fluid was collected at different time points post-infection, with three replicates at each time point. The virus was analyzed using TCID50. 50 The viral titer was determined at various time points, and a multi-step growth curve of the chimeric virus was plotted.

4. The method for identifying PEDV chimeric virus attenuation based on reverse genetics as described in claim 1, characterized in that, Step S7 also includes: In the pathogenicity test of piglets, dead piglets were necropsed, and piglets 7 days after challenge were euthanized and necropsed to observe gross pathological changes in the intestines; intestinal tissue samples were collected and pathological sections were prepared, and the tissue lesions were observed by HE staining.

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