Chimeric Newcastle disease virus as well as construction method and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a construction method and application of a chimeric Newcastle disease virus, the chimeric Newcastle disease virus is obtained by using the Newcastle disease virus as a vector to express an S protein of a porcine epidemic diarrhea virus variant, replacing a transmembrane region of the S protein with a transmembrane region of an F protein of the Newcastle disease virus, and obtaining the chimeric Newcastle disease virus. The intracellular region of the S protein is replaced with the intracellular region of the F protein of the Newcastle disease virus, and a new chimeric virus NDV SpottedDove-S / F is obtained by using a reverse genetic technology. The chimeric virus is safe to sows, induces the body to generate high-level PEDV specific antibodies and neutralizing antibodies, and can provide passive immune protection for newborn piglets, so that the chimeric virus NDV SpottedDove-S / F is expected to be used as a candidate vaccine for controlling high-pathogenicity PEDV variants.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, and in particular to a chimeric Newcastle disease virus and a construction method and application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious enteric disease of pigs caused by porcine epidemic diarrhea virus (PEDV). The main clinical symptoms are watery diarrhea, vomiting and dehydration. The morbidity rate of newborn piglets is 100%, and the mortality rate is 80%-100%. Since 2010, highly pathogenic PEDV variants have broken out in countries around the world, causing the death of a large number of piglets and posing a huge threat to the global pig industry. Therefore, it is urgent to develop a safe and effective vaccine to control the current epidemic of PEDV.

[0003] PEDV S protein is the largest membrane protein. The S protein of the attenuated vaccine strain PEDV CV777 is a type I glycoprotein composed of 1383 aa, including a short signal peptide region (1-24aa), an extracellular region (25-1323aa), a transmembrane domain (TM) (1324-1356aa) and a short intracellular region (CT) (1357-1383aa). S protein is the main immunogenic protein of PEDV, and multiple neutralizing epitope regions have been identified: S1 0 (aa 19–220), S1 A (aa435–485), COE (aa 499–638), SS2 (aa 748–755), SS6 (aa 764–771), 2C10 (aa1368–1374), and are also the main target proteins of viral vector vaccines, subunit vaccines, and mRNA vaccines. Multiple vectors such as adenovirus, aphthous ulcer virus, and vesicular stomatitis virus expressing PEDV S protein can induce pigs to produce high levels of neutralizing antibodies and provide protection against virus attack.

[0004] Coronaviruses escape the original immunity through continuous antigenic evolution, and this immune escape mutation often makes the pathogen more pathogenic. Several studies have confirmed that the attenuated vaccine PEDV CV777 cannot completely protect the current PEDV GⅡ epidemic strain, but some farms and farmers in my country still use this vaccine for immunization. In addition, the study found that the attenuated vaccine developed by the traditional cell-passage attenuation strategy for the mutant PEDV is safe for pigs, but it still cannot provide complete protection. This is mainly because PEDV undergoes adaptive mutations during cell passage, resulting in changes in its antigenicity. However, the inactivated PEDV vaccine developed by cell propagation also has this problem, and it is difficult for the protective antigen to exist stably. Therefore, the update of the new generation of vaccines needs to pay more attention to antigen stability while ensuring safety. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a chimeric Newcastle disease virus and a construction method and application thereof.

[0006] A chimeric Newcastle disease virus, wherein the chimeric Newcastle disease virus uses Newcastle disease virus (NDV) as a vector to express the S protein of a porcine epidemic diarrhea virus variant, the transmembrane region of the S protein is replaced by the transmembrane region of the F protein of the Newcastle disease virus, and the intracellular region of the S protein is replaced by the intracellular region of the F protein of the Newcastle disease virus, to obtain the chimeric Newcastle disease virus.

[0007] Preferably, the Newcastle disease virus is NDV SpottedDove / China / 08, and the porcine epidemic diarrhea virus variant is PEDV CH / Yinchuan / 2021 variant;

[0008] The NCBI number of the NDV SpottedDove / China / 08 is KC934170;

[0009] The NCBI number of the PEDV CH / Yinchuan / 2021 is OP480878.

[0010] A method for constructing a chimeric Newcastle disease virus comprises the following steps:

[0011] Primer 1 was used to amplify the extracellular region gene of the S protein of PEDV CH / Yinchuan / 2021, primer 2 was used to amplify the TM gene and CT gene fragments of the NDVSpottedDove F protein, and primer 3 was used to amplify the NDV SpottedDove M gene;

[0012] The S protein extracellular region gene, TM gene and CT gene fragments and NDV

[0013] The SpottedDove M gene was fused into a gene fragment S / F, and the S / F was cloned between the P and M genes of pT7-SpottedDove to obtain a plasmid;

[0014] The plasmid is used to obtain the chimeric Newcastle disease virus through reverse genetic operation technology.

[0015] Preferably, the sequence of the primer 1 is shown as SEQ ID NO.1-2, the sequence of the primer 2 is shown as SEQ ID NO.3-4, and the sequence of the primer 1 is shown as SEQ ID NO.5-6.

[0016] A vaccine comprises the chimeric Newcastle disease virus and pharmaceutically acceptable auxiliary materials.

[0017] Preferably, each animal receives ≥10 7 TCID 50 / mL.

[0018] Preferably, the pharmaceutically acceptable excipient is one or more of a buffer, a protective agent, a stabilizer, a surfactant and an osmotic pressure regulator.

[0019] The chimeric NDV is used in preparing a product for preventing porcine epidemic diarrhea virus.

[0020] Preferably, the vaccine contains the chimeric Newcastle disease virus as an active ingredient.

[0021] Preferably, the porcine epidemic diarrhea virus is PEDV CH / Yinchuan / 2021.

[0022] The present invention uses reverse genetic manipulation technology to express PEDV CH / Yinchuan / 2021S protein using the attenuated strain NDV SpottedDove / China / 08 (NDVSpottedDove for short) as a vector, and replaces the TM and CT of the S protein with the TM and CT of the NDV F protein to construct a chimeric virus NDV SpottedDove-S / F. The results of IFA, WB, and one-step growth curves show that NDVSpottedDove-S / F can normally express the S protein, and has no significant difference in growth kinetics with the parent strain NDV SpottedDove and the recombinant virus NDVSpottedDove-S.

[0023] In order to further evaluate whether NDV SpottedDove-S / F has genetic stability, the chimeric virus was passaged in chicken embryos for 30 generations, and each generation of virus was identified by RT-PCR and sequencing. The results showed that the inserted exogenous genes PEDV S and TM and CT genes of NDV F proteins could be detected in each generation of virus, and the genes did not mutate. In addition, IFA and WB further proved that NDV SpottedDove-S / F could stably express PEDV S protein during passage.

[0024] In order to evaluate the pathogenicity of NDV SpottedDove-S / F, the chimeric virus was inoculated into 9-day-old chicken embryos and 1-day-old SPF chickens, and the MDT and ICPI were determined. The results showed that the MDT and ICPI values ​​of NDV SpottedDove-S / F were 97.6 and 0.4, respectively, which were not significantly different from the MDT (96.64h) and ICPI (0.4) of the parental strain NDV SpottedDove. These data indicate that NDV SpottedDove-S / F can stably express the S protein, and the insertion of exogenous genes did not change its growth dynamics and pathogenicity.

[0025] The highly pathogenic PEDV strains currently prevalent in my country are clinically characterized by rapid onset, rapid spread, rapid death, and high mortality. In addition, enteric coronaviruses have a greater impact on newborn animals. In addition, the immune system of newborn piglets is not yet mature, so active immunization of newborn piglets cannot prevent PEDV infection at all. At present, milk-derived immunity is still the most promising and effective way to protect newborn suckling piglets from enteric pathogens such as PEDV. The key to this method is that the immune sows passively transfer sufficient sIgA antibodies to newborn piglets through the intestinal-mammary-sIgA axis through colostrum or normal milk.

[0026] The present invention immunizes the sows with the chimeric virus NDV SpottedDove-S / F through HA at 6, 4, and 2 weeks before delivery. After immunization, the sows are evaluated for safety, and the IgG, IgA and anti-PEDV neutralizing antibodies in the sow serum, colostrum and normal milk are measured. The results showed that no adverse clinical reactions were observed in the sows after immunization, including loss of appetite, fever, allergies, miscarriage, stillbirth and other conditions, and healthy piglets were delivered as scheduled according to the expected date of delivery. The antibody test results showed that the serum and colostrum of the immunized sows contained high levels of IgG, IgA and neutralizing antibodies, and there was no significant difference in the antibodies induced by sows immunized with inactivated PEDV CH / Yinchuan / 2021. These data prove that the chimeric virus NDV SpottedDove-S / F is safe for sows and can induce the body to produce high levels of PEDV-specific antibodies and neutralizing antibodies.

[0027] In order to evaluate the passive immune protection effect of NDV SpottedDove-S / F on suckling piglets, the blood of piglets before and after colostrum intake was collected to detect the level of maternal antibodies, and the piglets were orally challenged with highly pathogenic PEDV CH / Yinchuan / 2021 at 5dpf. In the previous NDV SpottedDove-S piglet passive immune protection experiment, the piglets were fed artificially. This challenge model made it difficult to ensure the natural growth conditions of piglets and sufficient milk intake, and the piglets failed to ingest milk containing maternal antibodies during the challenge period. In order to simulate the natural growth environment of piglets and ensure that piglets take in enough breast milk, the piglets in the NDV SpottedDove-S / F passive immune challenge experiment were raised separately from the sows in a farrowing bed, and the piglets were allowed to suck breast milk freely. The results showed that no IgG, IgA and neutralizing antibodies were detected in the serum of all piglets before ingestion of colostrum, while piglets in the NDV SpottedDove-S / F group obtained high levels of IgG, IgA and neutralizing antibodies at 3 and 5 dpf, and there was no significant difference with the PEDVCH / Yinchuan / 2021 group. These data further prove that newborn piglets can only obtain maternal antibodies through milk intake, and NDV SpottedDove-S / F immunized sows can passively transfer high levels of maternal antibodies to newborn suckling piglets through colostrum and normal milk. In addition, the results of the challenge experiment showed that the severity of diarrhea in piglets in the NDV SpottedDove-S / F group was significantly reduced compared with the control group, and the piglet survival rate was consistent with that of the PEDV CH / Yinchuan / 2021 group, both at 80%. The piglets also lost less weight, reduced detoxification, shortened detoxification time, and reduced intestinal tissue damage. These data prove that NDV SpottedDove-S / F can provide passive immune protection to piglets by alleviating diarrhea, reducing mortality, reducing detoxification, and reducing tissue damage.

[0028] In order to evaluate the clinical protective effect of PEDV CV777 vaccine against the current highly pathogenic PEDV, this study evaluated the passive immune protection of piglets by immunizing pregnant sows with PEDV CV777. The results showed that the serum and colostrum of sows immunized with PEDV CV777 and the serum of piglets contained high levels of IgG and IgA antibodies, which were not significantly different from the NDV SpottedDove-S / F and PEDV CH / Yinchuan / 2021 groups, but the neutralizing antibodies were significantly lower than those of the above two groups. The data from the challenge experiment showed that the diarrhea severity of piglets in the PEDVCV777 group was higher than that in the NDV SpottedDove-S / F and PEDV CH / Yinchuan / 2021 groups, the survival rate was lower (40%), the toxin excretion volume was higher, the toxin excretion period was longer, and the pathological changes in intestinal tissues were obvious. These data further prove that passive immunization with PEDVCV777 cannot provide effective protection against the current PEDV epidemic variants.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention uses reverse genetic manipulation technology to construct a chimeric Newcastle disease virus NDV SpottedDove-S / F expressing the S protein of a porcine epidemic diarrhea virus variant, and evaluates the protective effect of maternal antibodies on piglets against virus infection by immunizing pregnant sows, in order to make the chimeric virus NDV SpottedDove-S / F a candidate vaccine for controlling the current PEDV variants. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure shows the construction and identification of NDV SpottedDove-S / F, wherein A is a schematic diagram of the construction of the chimeric virus NDV SpottedDove-S / F, B is the IFA verification of the expression of the S protein, C is the WB verification of the expression of the S protein, and D is the one-step growth curve of NDV SpottedDove-S / F;

[0032] Figure 2 This is the identification of the genetic stability of NDV SpottedDove-S / F. A is the IFA verification of the expression of the S protein of NDV SpottedDove-S / F at P10, P20, and P30. B is the WB verification of the expression of the S protein of NDV SpottedDove-S / F at P10, P20, and P30.

[0033] Figure 3 are antibodies produced by sows induced by NDV SpottedDove-S / F, A is the design of the immune challenge experiment, B is the IgG antibody induced by sows after immune challenge, C is the IgA antibody induced by sows after immune challenge, and D is the PEDV neutralizing antibody level in the serum of sows after immune challenge; Note: The column descriptions in Figures C and D are the same as those in Figure B;

[0034] Figure 4 The antibodies in the breast milk of sows immunized with NDV SpottedDove-S / F, A is the IgG antibody in the breast milk of sows after farrowing, B is the IgA antibody in the breast milk of sows after farrowing, and C is the neutralizing antibody level in the breast milk of sows after farrowing; Note: The column descriptions in Figures B and C are the same as those in Figure A;

[0035] Figure 5 is the maternal antibody level of piglets, A is the IgG antibody in the serum of piglets after ingesting breast milk, B is the IgA antibody in the serum of piglets after ingesting breast milk, and C is the PEDV neutralizing antibody level in the serum of piglets after ingesting breast milk; Note: The column descriptions in Figures B and C are the same as those in Figure A;

[0036] Figure 6 Figure 2 is the passive immune protection of NDV SpottedDove-S / F on piglets, A is the clinical score of diarrhea after piglet challenge, B is the survival rate of piglet challenge, C is the weight of piglet challenge, and D is the amount of PEDV excreted after piglet challenge; Note: The line descriptions in Figures B, C, and D are the same as those in Figure A;

[0037] Figure 7 This is the passive immune protection of NDV SpottedDove-S / F on piglets. A shows the pathological changes in the intestinal tract of piglets after challenge with the virus. B shows the pathological changes in the intestinal tract of piglets after challenge with the virus by HE staining. DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0039] The sources of materials used in the present invention are as follows:

[0040] PEDV CH / Yinchuan / 2021 (NCBI Accession No.OP480878) and NDV SpottedDove / China / 08 (NDV SpottedDove) (NCBI Accession No.KC934170) were isolated and preserved by the Animal Infectious Disease Laboratory, College of Veterinary Medicine, Northwest A&F University. NDV SpottedDove-S was rescued and preserved by the Animal Infectious Disease Laboratory, College of Veterinary Medicine, Northwest A&F University. PEDV attenuated vaccine strain CV777 was preserved by the Animal Infectious Disease Laboratory, College of Veterinary Medicine, Northwest A&F University. 9-day-old SPF chicken embryos and SPF chicks were purchased from Shandong Haotai Experimental Animal Breeding Co., Ltd. pT7-SpottedDove (NDV SpottedDove / China / 08 plasmid containing the full-length gene of the T7 promoter), pT7-SpottedDove-S (the full-length gene of the PEDV CH / Yinchuan / 2021 strain S was inserted between the P and M genes of the pT7-SpottedDove plasmid), pCAGGs-T7 (plasmid expressing T7 RNA polymerase), and pCMV-NPL (helper plasmid expressing NDV LaSotaNP, P, and L genes) were constructed and maintained by the Animal Infectious Diseases Laboratory, College of Veterinary Medicine, Northwest A&F University.

[0041] DMEM, FBS, and trypsin (liquid and powder) were purchased from GIBCO; Trizol (RNAiso), endonuclease, high-fidelity enzyme Prime STAR MAX, T4 DNA ligase, and pMD19-T plasmid were purchased from Dalian Bao Biotechnology Co., Ltd.; reverse transcription kit (StarScript First-strand cDNA Synthesis Mix) and common PCR premix were purchased from GenStar; plasmid mini-extraction kit and gel recovery kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; primers were synthesized by Xi'an Qingke Jersey Company; NDV NP protein polyclonal antibody was prepared and stored by this laboratory. PEDV S1 protein monoclonal antibody was purchased from Medgene Laboratory, USA. Sheep anti-mouse IgG-HRP and β-Actin monoclonal antibody were purchased from Abcam; SYBR qPCR Master Mixture kit was purchased from Beijing Dining Biotechnology Co., Ltd. PEDV S protein IgG and IgA ELISA antibody detection kits were purchased from Shanghai Enzyme Biotechnology Co., Ltd. PVDF membrane was purchased from GE, USA.

[0042] Thirteen multiparous sows with the same due date were purchased from a farmer in Yangling Demonstration Zone, Shaanxi Province. RT-PCR and antibody tests determined that the sows had not been immunized with PEDV, PDCoV, PoRV, or TGEV vaccines or infected with the above pathogens. Animal experiments were conducted at the Experimental Animal Center of Northwest A&F University.

[0043] The experiments involved in the present invention were carried out according to the following steps, and all procedures are conventional methods unless otherwise specified.

[0044] IFA (Immunofluorescence assay)

[0045] Vero cells with good growth status were inoculated in 24-well plates. When the cells were full of monolayers, the culture medium was discarded. BHK-21 cells were inoculated with 0.1 MOI of NDV SpottedDove-S / F and NDV SpottedDove strains, and BHK-21 cells without any treatment were set as negative controls. After 36 hours of culture, the culture medium was discarded, and pre-cooled anhydrous ethanol was added to fix the cells for 30 minutes, and then the anhydrous ethanol was discarded. After washing the cells three times with PBS, the primary antibody (NDV NP protein polyclonal antibody 1:1000, PEDV S1 protein monoclonal antibody 1:1000) was added, and incubated in a 37°C incubator for 1 hour, and then 1:1000 diluted FITC-labeled goat anti-mouse IgG was added, incubated at 37°C for 1 hour, and washed with PBS 5 times, and the expression of PEDVS and NDVNP proteins was observed under a fluorescent inverted microscope.

[0046] WB (Western blot, protein immunoblotting)

[0047] BHK-21 cells were infected with NDV SpottedDove-S / F and NDV SpottedDove strains at a dose of 0.1 MOI, and the virus was harvested after 24 hours. Cells not inoculated with the virus were set as negative controls. The specific steps are as follows:

[0048] (1) Sample collection: 24 h after the virus infected Vero cells, discard the culture medium, add RIPA lysis buffer and lyse on ice for 30 min, stir with a pipette to completely lyse the cells, and transfer to a 1.5 mL EP tube;

[0049] (2) Sample cooking: dilute the lysed sample with 5× loading buffer and cook it in a 100°C metal bath for 5 min. Store it in a 4°C refrigerator until use.

[0050] (3) Gel preparation: Prepare the separation gel (lower gel) and replace the 1.5 M Tris-HCl at pH 8.8 with the concentrated gel at pH 6.8;

[0051] (4) Electrophoresis: Spot the prepared samples in the stacking gel, 10 μL / well, keep the stacking gel at a constant voltage of 80 V for 30 min, and keep the separation gel at a constant voltage of 120 V for 1-2 h. Stop the electrophoresis when the bromophenol blue electrophoresis reaches the lowest end.

[0052] (5) Transfer: constant current wet transfer, 100 mA for 1 h;

[0053] (6) Blocking: The PVDF membrane was rinsed with TBST three times, 5 min each time, and then the membrane was immersed in 10% skim milk powder and incubated overnight at 4°C;

[0054] (7) Incubation with primary antibody: The blocked PVDF membrane was rinsed with TBST five times, 10 min each time, and then the membrane was placed in the diluted primary antibody at 4°C overnight;

[0055] (8) Incubation with secondary antibody: After incubation with primary antibody, the PVDF membrane was rinsed with TBST 5 times, 10 min each time, and then the membrane was incubated in the diluted secondary antibody at room temperature for 1 h;

[0056] (9) Exposure: After incubation with secondary antibodies, the PVDF membrane was rinsed with TBST five times, each time for 10 min. Finally, the membrane was exposed on a protein exposure instrument, and the image was analyzed and saved.

[0057] Determination of virus titer and growth curve

[0058] BHK-21 cells were seeded in a 96-well plate at 100 μL / well and cultured in a 37°C, 5% CO2 cell culture incubator for 24 h. The culture medium was discarded when the cells grew into a monolayer. Then NDV SpottedDove-S / F was diluted 10 times and added to a 96-well plate at 100 μL / well and cultured in a 37°C, 5% CO2 cell culture incubator for 72 h. The infected virus was then determined by IFA, and the titer was calculated according to the Reed and Muench method. TCID 50 .

[0059] In order to determine the growth dynamics of NDV SpottedDove-S / F, the toxicity of the recombinant virus and the parental strain NDVSpottedDove at different time points was measured, and a one-step growth curve was drawn. BHK-21 cells in good growth state were inoculated in a six-well plate. When the cells were covered with a monolayer, BHK-21 cells were infected with 0.1MOI doses of NDV SpottedDove-S / F and NDVSpottedDove strains. The virus was harvested every 8 hours, and the virus harvested at each time point was frozen and thawed three times. The virus TCID at each time point was determined according to the above method for determining the virus toxicity. 50 , repeated three times, growth curves were drawn and the differences in growth kinetics between SpottedDove-S and NDV SpottedDove were analyzed.

[0060] Immunity challenge experiment

[0061] Anal swabs and serum were collected from 13 sows with the same due date before the experiment. PEDV, PDCoV, PoRV, and TGEV nucleic acid and antibody were determined to be negative by RT-PCR and antibody detection. PEDV CH / Yinchuan / 2021 was inactivated with 4% formaldehyde solution and stored for later use. All sows were randomly divided into 5 groups: NDV SpottedDove-S / F (n=3), PEDV CH / Yinchuan / 2021 (n=3), PEDV CV777 (n=3), NDV SpottedDove (n=2), and DMEM (n=2). They were immunized with HA in 3 mL of NDV SpottedDove-S / F (10 7 TCID 50 / mL), inactivated PEDV CH / Yinchuan / 2021, PEDVCV777(10 6 TCID 50 / mL)、NDV SpottedDove(10 7 TCID 50 / mL) and DMEM. The same dose and immunization route were used to immunize sows three times 6, 4, and 2 weeks before delivery. After immunization, the sows' clinical symptoms and appetite were observed every day, rectal temperature was measured, and health status and expected delivery date were assessed. Sow colostrum was collected within 12 hours after delivery, and normal milk was collected 3 and 5 days after delivery (dpf, days post-farrowing), respectively. In order to simulate the natural growth conditions of lactating piglets, piglets after delivery were allowed to suck breast milk freely during the entire experimental period, including the period after the virus attack. Ten healthy piglets were selected from the piglets born by sows in each group and raised separately with one of the sows on a separate delivery bed. Two groups were selected from the DMEM group, one group was the virus attack group DMEM, and the other group was the control group Mock. At 5dpf, all piglets except the Mock group were orally challenged with 1mL of the virulent strain PEDV CH / Yinchuan / 2021 (10 4.5 TCID 50 / mL). All piglets in the Mock group were fed 1mL DMEM as a placebo. The clinical symptoms of the piglets, including diarrhea, vomiting, body weight and mortality, were monitored every day after the challenge, and the experiment ended at 15dpc.

[0062] The severity of diarrhea was assessed by the consistency of feces in the diarrheal pigs, and the scoring criteria were as follows: 0: normal feces; 1: semi-dry feces; 2: very soft feces, close to liquid; 3: watery diarrhea with some solids; 4: watery diarrhea without solids. Rectal swabs were collected every day until the end of the experiment. During the experiment, the dead piglets were autopsied and analyzed for visual pathology. At the same time, the duodenum, jejunum, and ileum tissues were collected and fixed with 4% formaldehyde solution to prepare pathological sections. At the end of the experiment, all surviving pigs were euthanized.

[0063] Sample processing and RNA extraction

[0064] Fecal swabs collected at different time points were diluted with 1 mL of sterile PBS, centrifuged at 4500 × g for 10 min at 4°C, and 300 μL of the supernatant was used for RNA extraction. For tissue samples, an equal amount of 0.1 g of tissue was weighed and homogenized with a grinder, then diluted with 1 mL of sterile PBS, centrifuged at 4500 × g for 20 min at 4°C, and 300 μL of the supernatant was used for RNA extraction. The specific steps are as follows:

[0065] (1) Add 700 μL RNAiso Plus reagent to 300 μL of the prepared stool or tissue sample, mix by inverting, and let stand at room temperature for 10 min.

[0066] (2) Add 200 μL of chloroform and shake on an oscillator for 10 seconds to mix thoroughly, then let stand at room temperature for 10 minutes;

[0067] (3) Centrifugation at 12,000 × g for 10 min at 4°C;

[0068] (4) Take a 1.5 mL EP tube without RNase and gently transfer the supernatant after centrifugation to the EP tube to avoid turbidity being sucked into the middle layer and the lower organic phase;

[0069] (5) Add an equal volume of pre-cooled isopropanol, gently invert the mixture to mix thoroughly, and place in a -20°C refrigerator for 30 min;

[0070] (6) Centrifugation at 12,000 × g for 15 min at 4°C;

[0071] (7) Discard the supernatant, add pre-cooled 75% ethanol, and gently blow the bottom precipitate with a pipette to mix it thoroughly;

[0072] (8) Centrifugation at 7500 × g for 5 min at 4°C;

[0073] (9) Gently discard the supernatant, invert the EP tube on absorbent paper, dry at room temperature for 15 min, add 20-50 μL RNase-free water to dissolve the precipitate, and extract the RNA for the next step of reverse transcription or store in a -80°C refrigerator for later use.

[0074] Reverse transcription

[0075] According to the instructions of the StarScript First-strand cDNA Synthesis Mix kit, reverse transcribe the sample. Before reverse transcription, the RNA concentration needs to be measured to meet the reverse transcription requirements. The specific operation is as follows:

[0076] (1) System preparation

[0077] In order to avoid RNA degradation, all operations were performed on an ice box when preparing the system. The total volume was 20 μL. The system was as follows:

[0078] Table 1 Reverse transcription system

[0079] Element volume Total RNA 2μL Oligo(dT)18(50μM) 1μL 2×Reactionmix 10μL StarScriptRTMix 1μL <![CDATA[RNase-FreeddH2O]]> 6μL

[0080] (2) Reaction procedure

[0081] The prepared samples were mixed by oscillation and centrifuged briefly, and reverse transcription was performed according to the following procedures: 42°C, 30 min; 85°C, 5 min. The synthesized cDNA samples were used for subsequent experiments or stored at -20°C for future use.

[0082] Fluorescence quantitative RT-PCR and establishment of standard curve

[0083] (1) Fluorescence quantitative RT-PCR

[0084] Fluorescent quantitative primers were designed by targeting the PEDV M gene. The primers are as follows: PEDV-qPCR-F: 5′-TATAAGGTTGCTACTGGCGT-3′, recorded as SEQ ID NO.7; PEDV-qPCR-R: 5′-CATTGACTGAACGACCAACA-3′, recorded as SEQ ID NO.8. The operation was performed according to the instructions of the SYBR qPCRMaster Mixture kit. The reaction system is as follows:

[0085] Table 2 RT-PCR reaction system

[0086] Element volume cDNA 1μL Upstream primer 0.5μL Downstream primer 0.5μL 2×RealStarGreenFastMixture 10μL <![CDATA[RNase-FreeddH2O]]> 8μL Total 20μL

[0087] The above system samples were added into eight-tube PCR tubes, and three replicates were set. After instant centrifugation, the reaction was carried out in a fluorescence quantitative instrument. The procedure was as follows: pre-denaturation at 95°C for 2 min; 95°C for 15 s; 60°C for 30 s; and 45 cycles.

[0088] (2) Establishment of standard curve

[0089] PEDV CH / Yinchuan / 2021 was amplified using primers targeting the PEDV M gene to obtain the PCR target fragment, which was then cloned into the pMD19-T vector after gel recovery and transformed into the competent E. coli DH5α. Positive colonies were selected for mass culture. After the bacterial solution was correctly identified, the plasmid was extracted. After PCR and sequencing were performed again, the plasmid was named pMD19-TM. The copy number of the plasmid standard was calculated according to the following formula: [Copies = (mass / molecular weight) × 6.02 × 10 23 ]. Take the positive plasmid with OD260 / OD280 of 1.8-2.0 as the standard, dilute the pMD19-TM plasmid 10 times, and use the diluted plasmid as the template to perform fluorescence quantitative PCR according to the reaction system and procedure recommended in the SYBR qPCR Master Mixture kit manual. 1 -10 9 conpies / μL positive plasmid to establish a standard curve. After the virus attack, anal swabs were collected from all pigs, and the PEDV excretion was evaluated by fluorescent quantitative RT-PCR.

[0090] ELISA

[0091] After immunization, IgG and IgA specific antibodies of PEDV S protein in sow colostrum, milk, serum and piglet serum were detected using commercial ELISA detection kits. The specific operation steps are as follows:

[0092] (1) Take out the required strips from the aluminum foil bag and equilibrate them at room temperature for 20 min. Seal the remaining strips in a ziplock bag and place them back at 4°C.

[0093] (2) Set up negative control wells, positive control wells and sample wells. Add 50 μL of control substance to each of the negative and positive control wells, add 50 μL of the sample to be tested to the sample well, and do not add any substance to the blank wells;

[0094] (3) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the control well and sample well, seal the wells with a sealing film, and incubate in a 37°C water bath or incubator for 60 min;

[0095] (4) Discard the liquid and pat dry on absorbent paper. Fill each well with washing solution (350 μL), let stand for 1 min, discard the washing solution, pat dry on absorbent paper, and repeat this process 5 times (you can also use a plate washer to wash the plate).

[0096] (5) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min;

[0097] (6) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min;

[0098] (7) Calculation of test results. The negative control OD value is less than 0.2, and the positive control OD value is greater than 0.8; Positive judgment (Cut-Off): The negative control OD value + 0.25, the sample OD value is greater than the threshold value and is judged as positive, otherwise, it is negative. Repeatability: The intra-plate coefficient of variation is less than 15%.

[0099] Virus neutralization assay

[0100] After immunization, neutralizing antibodies in sow colostrum, milk, serum and piglet serum were detected. The specific steps are as follows:

[0101] (1) Inactivate colostrum, milk, and serum samples in a water bath at 56°C for 30 min;

[0102] (2) The inactivated sample was serially diluted 2-fold (2-1024), and each dilution of the sample (100 μL) was mixed with PEDV CH / Yinchuan / 2021 (200 TCID 50 ) were mixed in equal volumes and incubated in a CO2 cell culture incubator for 1 h;

[0103] (3) The serum-virus mixture (100 μL) was transferred to a Vero cell culture medium (96-well plate) in good growth condition, incubated in a CO2 cell culture incubator for 1 h, then the mixture was discarded and the Vero cells were washed 3 times with DMEM.

[0104] (4) Add 100 μL of DMEM (containing 10 μg / mL trypsin) to the cells and culture them in a 37°C, 5% CO2 incubator for 48 h. Observe CPE under a microscope or perform IFA to determine the neutralization titer. Serum samples were tested three times.

[0105] (5) Neutralizing antibody titers were expressed as the reciprocal of the highest dilution without CPE in all replicate wells.

[0106] Histopathology

[0107] Dead pigs and euthanized pigs were autopsied, and all tissues and organs were analyzed for gross pathological changes. Fresh intestinal tissues were fixed with 4% formaldehyde solution and sent to Shaanxi Yike Biotechnology Service Co., Ltd. for preparation of pathological sections.

[0108] Statistical analysis

[0109] Data were analyzed by variance analysis and plotted using GraphPad Prism 9.4.1 software. Paired t-test was used for comparison between two groups. One-way ANOVA and t-test were used for multiple group comparisons. Kaplan-Meier method was used for survival analysis. P<0.05(*), P<0.01(**), P<0.001(***), P<0.0001(****).

[0110] Example 1

[0111] Rescue and identification of chimeric virus NDV SpottedDove-S / F

[0112] To construct the chimeric virus NDV SpottedDove-S / F, the TM and CT genes of the S protein of the PEDV CH / Yinchuan / 2021 strain were replaced with the TM and CT of the F protein of the NDV SpottedDove strain and cloned between the P and M genes of pT7-SpottedDove to construct the pT7-SpottedDove-S / F plasmid ( Figure 1 A), as follows:

[0113] According to the primers designed in Table 3, the S protein extracellular region gene containing PEDV CH / Yinchuan / 2021 was amplified using primers PEDV-S-Ectodomain with plasmid pT7-SpottedDove-S as a template; the TM and CT gene fragments of the NDV SpottedDove F protein were amplified using primers NDV-F-TM / CT with plasmid pT7-SpottedDove as a template; the gene fragment including the NDV SpottedDove M gene and restriction site was amplified using primers Dove-M with plasmid pT7-SpottedDove-S as a template; the above three gene fragments were fused into a gene fragment S / F by fusion PCR and cloned into the pMD19-T plasmid. The correctly identified plasmid was named pMD19-TS / F by further PCR and sequencing, and saved for later use.

[0114] Using plasmid pMD19-TS / F as a template, the fusion fragment S / F was amplified using primers PEDV-S-Ectodomain-F and Dove-MR designed in Table 3. After gel recovery, double restriction enzyme digestion (Pac I and AatII) was performed and then cloned between the P and M genes of the pT7-SpottedDove plasmid. The plasmid with the correct sequence was named pT7-SpottedDove-S / F and stored for future use.

[0115] The reverse genetics method for rescuing NDV was used to co-transfect plasmids pT7-SpottedDove-S / F, pCMV-NPL, and pCAGGs-T7 into BHK-21 cells to rescue the chimeric virus. CPE was observed after 48 hours to obtain the successfully rescued chimeric virus. The details are as follows:

[0116] Referring to the instructions of the transfection reagent Lipofectamine3000, the plasmids pT7-SpottedDove-S / F (2 μg), pCMV-NPL (2 μg), and pCAGGs-T7 (1 μg) were co-transfected into BHK-21 cells. After 48 hours, the cells were observed under an inverted fluorescence microscope for cytopathic effect (CPE). After 72 hours, the cells were scraped off with a pipette tip and collected with the supernatant in a 1.5 mL EP tube and stored in a -80°C refrigerator for later use.

[0117] The cell fluid showing CPE was collected and inoculated into 9-day-old SPF chicken embryos for further propagation. After 72 hours, the allantoic fluid was collected and preserved, and the presence of the PEDV S gene was identified by PCR and sequencing.

[0118] The collected cell and virus mixture was frozen and thawed three times and then centrifuged. 50 μL of the supernatant was inoculated into 9-day-old chicken embryos. The chicken embryos were then cultured in an incubator for 96 hours, and the allantoic fluid was collected in an EP tube and stored in a -80°C refrigerator for later use. In addition, the collected allantoic fluid was filtered and sterilized and inoculated into BHK-21 cells. CPE was observed under a microscope every 24 hours, and the successfully rescued NDV SpottedDove-S / F virus was identified by PCR, sequencing, immunofluorescence assay (IFA) and protein immunoblot (WB).

[0119] In addition, the collected viruses were inoculated into BHK-21 cells, and the expression of the chimeric virus NDVSpottedDove-S / FS protein was further identified by IFA.

[0120] In order to evaluate the growth characteristics of the chimeric virus NDV SpottedDove-S / F, it was inoculated into BHK-21 cells and a one-step growth curve was constructed.

[0121] Table 3 Primers used in the present invention

[0122]

[0123] result

[0124] The schematic diagram of constructing pT7-SpottedDove-S / F plasmid is as follows Figure 1 As shown in A, the identification results of the rescued NDV SpottedDove-S / F virus are as follows Figure 1 As shown in B-1D, CPE infected with NDV SpottedDove-S / F showed two fluorescent signals, red (anti-PEDV-S1 mouse monoclonal antibody) and green (anti-NDV-NP rabbit polyclonal antibody), and the two fluorescent signals were co-localized ( Figure 1 B). However, CPE in NDVSpottedDove-infected cells only showed a green fluorescence (anti-NDV-NP rabbit polyclonal antibody), while no CPE or fluorescence signal was observed in Mock-infected cells ( Figure 1 B). Meanwhile, WB further confirmed that the CPE in cells infected with NDV SpottedDove-S / F expressed both S and NP proteins, the parent strain NDV SpottedDove only expressed NP protein, and the expression of S and NP proteins was not detected in cells infected with Mock ( Figure 1 B).

[0125] One-step growth curve Figure 1As shown in D, the growth kinetics of NDV SpottedDove-S / F had no significant difference from that of the parental strain NDVSpottedDove and the recombinant virus NDV SpottedDove-S at 12-72 hpi, and the titers of the three viruses all reached peak at 48 hpi.

[0126] Example 2

[0127] Genetic stability and pathogenicity of NDV SpottedDove-S / F

[0128] To identify the genetic stability of NDV SpottedDove-S / F, the chimeric virus was passaged in 9-day-old chicken embryos for 30 generations, and the S gene of each generation of virus was identified by RT-PCR and sequencing.

[0129] In order to identify whether the S gene of the chimeric virus NDV SpottedDove-S / F is expressed normally during the passage process, the 10th, 20th, and 30th passage viruses were selected to inoculate BHK-21 cells, and the S protein expression was identified by IFA and WB.

[0130] In order to evaluate the pathogenicity of NDV SpottedDove-S / F, the chimeric virus was inoculated into 9-day-old chicken embryos and 1-day-old SPF chickens, and the MDT (mean embryo lethal time) and ICPI (chicken brain pathogenicity index) were measured. The MDT operation was as follows: the collected virus allantoic fluid was diluted 10 times with sterile PBS to 10 -5 -10 -9 , 5 9-day-old SPF chicken embryos were inoculated through the allantoic cavity for each dilution concentration, and the inoculation amount for each chicken embryo was 100μL, and they were cultured at 37℃. After an interval of 8h, another 5 chicken embryos were inoculated for each dilution, 100μL / embryo, and cultured at 37℃. The eggs were illuminated twice a day, and the observation was continued for 7 days, and the death time of each chicken embryo was recorded. The MDT value was calculated according to the following formula: MDT = (number of dead embryos at X hours × X hours + number of dead embryos at Y hours × Y hours) / total number of dead embryos. The ICPI operation is as follows: the virus fresh allantoic fluid is diluted 10 times with sterile PBS. One-day-old chicks were inoculated intracerebrally, and 10 chicks were inoculated with each virus, with a dosage of 50μL / chicken. The inoculated chicks were placed in an isolator for feeding and observed every 24h. The observation results were scored according to the standard, with normal chickens scored as 0 points, diseased chickens scored as 1 point, and dead chickens scored as 2 points. The observation lasted for a total of 10 days. The ICPI value was calculated according to the following formula: ICPI = (cumulative number of cases in 10 days × 1 + cumulative number of deaths in 10 days × 2) / cumulative total number of chickens in 10 days.

[0131] result

[0132] The CPE of cells infected with NDV SpottedDove-S / F at the 10th, 20th, and 30th generations showed red (anti-PEDV-S1 mouse monoclonal antibody) and green (anti-NDV-NP rabbit polyclonal antibody), and the two fluorescences were co-localized, while the CPE of cells infected with the parental strain NDVSpottedDove showed only green fluorescence, and the cells infected with Mock showed no CPE and fluorescence signal ( Figure 2 A). At the same time, WB results showed that NDV SpottedDove-S / F could also express S protein normally at the 10th, 20th and 30th generations ( Figure 2 B). The PEDV S gene and the TM and CT genes of NDV SpottedDove F were detected in each generation of virus, and the sequencing results also showed that the inserted genes did not undergo gene mutations. The pathogenicity results showed that the MDT and ICPI values ​​of NDV SpottedDove-S / F were 97.6 and 0.4, respectively.

[0133] Example 3

[0134] NDV SpottedDove-S / F induces antibodies in sows

[0135] To evaluate the passive immune protection of chimeric virus NDV SpottedDove-S / F on newborn piglets, sows were immunized with NDV SpottedDove-S / F, inactivated PEDV CH / Yinchuan / 2021 and live attenuated vaccine PEDV CV777 via HA route at 6, 4 and 2 weeks before farrowing. Figure 3 A.

[0136] After immunization, the safety of sows was evaluated and the IgG, IgA and anti-PEDV neutralizing antibodies in the sow serum were measured. The results are shown in Figure 3 .

[0137] result

[0138] The results showed that no adverse clinical reactions were observed in the sows in each group after immunization, including loss of appetite, fever, allergies, miscarriage, stillbirth, etc., and healthy piglets were born on schedule according to the expected date.

[0139] The results are as follows Figure 3As shown, the antibodies in the serum of all sows in the NDV SpottedDove-S / F group turned positive at 28dpi, and the antibody titer increased significantly after the second booster immunization (3B). Before the challenge (42dpi), the IgG antibody in the NDV SpottedDove-S / F group was significantly higher than that in the vehicle group (NDV SpottedDove group) and the control group (DMEM group) (P<0.0001), but there was no significant difference with the PEDV CH / Yinchuan / 2021 and PEDV CV777 groups (3B). The trend of IgA antibody levels in the serum of sows after immunization was basically consistent with that of IgG antibodies. Before the challenge (42dpi), NDV SpottedDove-S / F immunized sows induced high levels of IgA antibodies, which was not significantly different from the PEDV CH / Yinchuan / 2021 and PEDV CV777 groups (3C). In addition, there was no significant difference in the IgA antibody level between the PEDV CV777 group and the PEDV CH / Yinchuan / 2021 group before challenge (3C).

[0140] The results of neutralizing antibody assay showed that the NDV SpottedDove-S / F, PEDV CH / Yinchuan / 2021 and PEDVCV777 groups induced low levels of neutralizing antibodies at 14 dpi, and the antibody levels increased significantly after booster immunization (3D). The NDVSpottedDove-S / F group produced high levels of neutralizing antibodies (170.70±73.90) before challenge (42 dpi), which was not significantly different from the PEDVCH / Yinchuan / 2021 group (3D). It is worth noting that the IgG and IgA antibodies in the PEDV CV777 group were not significantly different from those in the NDVSpottedDove-S / F and PEDV CH / Yinchuan / 2021 groups at 28, 42 and 56 dpi, but the neutralizing antibodies were significantly lower than those in the above two groups after immunization and challenge (P<0.05).

[0141] Example 4

[0142] Antibodies in sow milk after immunization with NDV SpottedDove-S / F

[0143] To determine the antibody levels in sow milk after immunization with NDV SpottedDove-S / F, colostrum and milk were collected on days 0, 3, and 5 after farrowing to detect IgG, IgA, and neutralizing antibodies against PEDV. Figure 4 .

[0144] result

[0145] The results showed that the colostrum of sows immunized with NDV SpottedDove-S / F contained high levels of IgG and IgA antibodies, which were not significantly different from those in the colostrum of sows immunized with PEDV CH / Yinchuan / 2021 and PEDV CV777 groups ( Figure 4 A and B). In addition, it was found that the IgG level in normal milk at 3 and 5 dpf was significantly lower than that in colostrum, but normal milk contained a higher level of IgA antibodies ( Figure 4 A and B). Neutralizing antibody test results showed that the colostrum of the NDV SpottedDove-S / F group contained high levels of neutralizing antibodies (170.70±73.90), and there was no significant difference between the NDV SpottedDove-S / F group and the PEDV CH / Yinchuan / 2021 group at 0, 3, and 5 dpf ( Figure 4 C). At the same time, the results showed that the neutralizing antibodies in colostrum and milk of PEDV CV777-immunized sows were significantly lower than those in PEDV CH / Yinchuan / 2021 and NDV SpottedDove-S / F groups (P<0.01) ( Figure 4 C).

[0146] Example 5

[0147] Maternal antibody levels in piglets

[0148] In order to determine the level of maternal antibodies acquired by newborn piglets from breast milk, blood samples were collected from piglets before and after colostrum ingestion to test IgG, IgA and neutralizing antibodies. The results are shown in Figure 5 .

[0149] result

[0150] The results are as follows Figure 5 As shown in Figure 2, no IgG, IgA, or neutralizing antibodies were detected in all piglets before colostrum ingestion (0 dpf). Figure 5 A, B, and C). Piglets in the NDV SpottedDove-S / F group produced high levels of IgG and IgA antibodies after ingesting colostrum and milk (3 and 5 dpf), which was not significantly different from the PEDV CH / Yinchuan / 2021 and PEDV CV777 groups ( Figure 5 A and B). In addition, high levels of neutralizing antibodies were detected in piglets of the NDV SpottedDove-S / F group after ingesting colostrum and milk, and there was no significant difference between the NDVCH / Yinchuan / 2021 group ( Figure 5 C). However, at 3 and 5 dpf, the ability of the serum of PEDV CV777 group piglets to neutralize PEDV CH / Yinchuan / 2021 was significantly lower than that of NDV SpottedDove-S / F and PEDV CH / Yinchuan / 2021 groups (P<0.0001) ( Figure 5 C).

[0151] Example 6

[0152] Passive immune protection of piglets by NDV SpottedDove-S / F

[0153] In order to evaluate the passive protective effect of the chimeric virus NDV SpottedDove-S / F on suckling piglets, all piglets except the Mock group were orally challenged with highly pathogenic PEDV CH / Yinchuan / 2021 at 5dpf. In order to simulate the natural growth environment of piglets and ensure that piglets consume enough breast milk, the piglets and sows were raised separately in a farrowing bed throughout the experiment, and the piglets were allowed to suck breast milk freely.

[0154] All piglets in the NDV SpottedDove and DMEM groups began to show typical PED clinical symptoms at 1 dpc, including watery diarrhea, vomiting, dehydration, and huddling; severe diarrhea and dehydration occurred at 2-6 dpc, and the surviving piglets still had mild diarrhea at 7 dpc, which lasted until 12 dpc ( Figure 6 A). Piglets in the NDV SpottedDove-S / F group developed mild diarrhea at 1dpc, only 1 / 10 piglets developed severe diarrhea at 2-3dpc, and the rest of the piglets recovered to normal at 7dpc ( Figure 6 A). Piglets in the PEDV CH / Yinchuan / 2021 group also developed mild diarrhea on 1 dpc, of which 2 / 10 piglets developed severe diarrhea on 2-3 dpc, but the rest of the piglets recovered to normal on the 6th day of farrowing ( Figure 6 A). It is noteworthy that 7 / 10 piglets in the PEDV CV777 group had severe diarrhea, and the surviving piglets recovered to normal at 12dpc ( Figure 6 A). In the NDV SpottedDove-S / F group, one piglet died at 2 and 3 dpc, respectively, with a survival rate of 80%. In the PEDV CH / Yinchuan / 2021 group, one piglet died at 5 and 6 dpc, respectively, with a survival rate of 80%. Figure 6 B). The survival rate of piglets in the PEDV CV777 group was 40%, the survival rate of piglets in the NDV SpottedDove and DMEM groups was 20%, and no piglets died in the Mock group ( Figure 6 B).

[0155] The results of body weight measurement showed that the weight of piglets in the NDV SpottedDove-S / F group decreased slightly from 2 to 6 dpc, and began to increase gradually from 7 dpc. At 14 dpc, the weight was comparable to that of piglets in the Mock group ( Figure 6C). The weight trend of piglets in the PEDV CH / Yinchuan / 2021 group was basically the same as that in the NDV SpottedDove-S / F group. After the weight loss at 2-6 dpc, it gradually recovered and was comparable to the Mock group at 10 dpc ( Figure 6 C). However, the weight of piglets in the NDV SpottedDove and DMEM groups began to decrease at 1 dpc, and decreased significantly at 2-5 dpc. The weight trend of surviving piglets in the PEDV CV777 group was basically the same as that in the NDV SpottedDove and DMEM groups. In addition, the results showed that although there were surviving piglets in the PEDV CV777, NDV SpottedDove and DMEM groups after the virus attack, their weight gain was very slow, and some piglets had zero weight gain for 3 consecutive days ( Figure 6 C).

[0156] Fecal swabs were collected from piglets every day after the infection and PEDV excretion was detected by qRT-PCR. The results showed that low-copy PEDV RNA (P276 RNA) was detected in all infected piglets except the Mock group at 1 dpc. Figure 6 D). Low-copy virus was detected in the NDV SpottedDove-S / F group at 1-6 dpc, and no viral RNA was detected in the feces of piglets at 7 dpc. The amount and cycle of virus excretion in piglets in the PEDV CH / Yinchuan / 2021 group were basically consistent with those in the NDV SpottedDove-S / F group, and virus excretion stopped at 6 dpc ( Figure 6 D). In contrast, high copies of viral RNA (8.49-8.55log 10 copies / μL), and low-copy virus can still be detected in feces at 14dpc ( Figure 6 D). In addition, the results showed that the surviving piglets in the PEDV CV777 group continued to excrete virus until 11dpc ( Figure 6 D).

[0157] The results of pathological examination showed that the NDV SpottedDove group and the DMEM group had obvious intestinal lesions, including severe congestion and blackening of the mesentery, intestinal adhesion, and loss of intestinal elasticity ( Figure 7 A). The intestines of piglets in the PEDV CV777 group also showed obvious pathological changes (swelling and transparent, severe intestinal adhesions and filled with yellow fluid), especially in the small intestine ( Figure 7 A). In contrast, no obvious ocular pathological changes were observed in the Mock, NDV SpottedDove-S / F, and PEDV CH / Yinchuan / 2021 groups ( Figure 7 A).

[0158] Histopathological findings showed that the duodenum, ileum, and ileum of piglets in the PEDV CV777, NDV SpottedDove, and DMEM groups showed severe epithelial cell loss, villous atrophy, and damage, while no obvious histopathological changes were observed in the Mock, NDV SpottedDove-S / F, and PEDVCH / Yinchuan / 2021 groups ( Figure 7 B).

[0159] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0160] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0161] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A chimeric Newcastle disease virus, characterized in that The chimeric Newcastle disease virus uses the Newcastle disease virus as a vector to express the S protein of a variant strain of porcine epidemic diarrhea virus, replaces the transmembrane region of the S protein with the transmembrane region of the F protein of the Newcastle disease virus, and replaces the intracellular region of the S protein with the intracellular region of the F protein of the Newcastle disease virus, and obtains the chimeric Newcastle disease virus using reverse genetics technology.

2. The chimeric Newcastle disease virus according to claim 1, characterized in that The Newcastle disease virus is NDVSpottedDove / China / 08, and the porcine epidemic diarrhea virus variant is the PEDV CH / Yinchuan / 2021 variant; The NCBI number of the NDV SpottedDove / China / 08 is KC934170; The NCBI number of the PEDV CH / Yinchuan / 2021 is OP480878.

3. A method for constructing a chimeric Newcastle disease virus according to claim 2, characterized in that: The following steps are involved: Primer 1 was used to amplify the extracellular region gene of the S protein of PEDV CH / Yinchuan / 2021, primer 2 was used to amplify the TM gene and CT gene fragments of the NDVSpottedDove F protein, and primer 3 was used to amplify the NDV SpottedDove M gene; The S protein extracellular region gene, TM gene and CT gene fragments and NDV The SpottedDove M gene was fused into a gene fragment S / F, and the S / F was cloned between the P and M genes of pT7-SpottedDove to obtain a plasmid; The plasmid is used to obtain the chimeric Newcastle disease virus through reverse genetic operation technology.

4. The construction method according to claim 3, characterized in that: The sequence of the primer 1 is shown as SEQ ID NO.1-2, the sequence of the primer 2 is shown as SEQ ID NO.3-4, and the sequence of the primer 1 is shown as SEQ ID NO.5-6.

5. A vaccine, characterized in that The vaccine comprises the chimeric Newcastle disease virus according to any one of claims 1 to 2 and pharmaceutically acceptable adjuvants.

6. The vaccine according to claim 5, characterized in that Each animal receives ≥ 10 7 TCID 50 / mL.

7. The vaccine according to claim 5, characterized in that The pharmaceutically acceptable excipients are one or more of a buffer, a protective agent, a stabilizer, a surfactant and an osmotic pressure regulator.

8. Use of the chimeric Newcastle disease virus according to claim 1 in the preparation of a product for preventing porcine epidemic diarrhea virus.

9. The use according to claim 8, characterized in that: The product is a vaccine, which uses the chimeric virus as an active ingredient.

10. The use according to claim 8, characterized in that: The variant strain of porcine epidemic diarrhea virus is the PEDVCH / Yinchuan / 2021 variant strain.

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