GIIa type porcine epidemic diarrhea virus strain and application thereof

Abstract

The application relates to the technical field of animal virology, and particularly discloses a GIIa type porcine epidemic diarrhea virus (PEDV) strain and application. The PEDV strain is named as LZ202401, and was preserved in the China Center for Type Culture Collection on October 15, 2024, with a preservation number of CCTCC NO:V202488. The strain provided by the application is a strong pathogenic PEDV strain, and after infecting piglets, the piglets appear vomiting and diarrhea conditions, so that the piglets are relatively slow in development, rough in hair, seriously dehydrated, depressed in spirit and constantly tremble. The inactivated vaccine prepared by using the strain has good immunogenicity and can be used for preventing porcine epidemic diarrhea caused by PEDV.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal virology, and particularly relates to a GIIa type porcine epidemic diarrhea virus strain and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is a highly susceptible contact infectious disease of piglets caused by porcine epidemic diarrhea virus (PEDV), which can cause piglets to have acute and dehydrating diarrhea as the main clinical features. The porcine epidemic diarrhea virus (PEDV) belongs to the members of the order Nestivirus, the family Coronaviridae and the genus alphacoronavirus, and has a spherical shape with a diameter of 95-190 nm. The virus has typical morphological characteristics of coronaviruses, and the virus is coated with a capsid membrane, and the capsid membrane has relatively standardized and radial spicules. The virus is a single-stranded RNA virus, and the full-length genome is about 28 kb, which contains 5' non-coding region, 3' non-coding region (Untranslated regions, UTR) and 7 open reading frames (Open reading frame, ORF). The 7 open reading frames encode 4 structural proteins, respectively, which are spicule protein (S protein), membrane protein (M protein), envelope protein (E protein) and nucleocapsid protein (N protein); a group of non-structural proteins (Nsp1-16) is produced by the cleavage products of ORF1a and ORF1b polypeptides; in addition, ORF3 encodes an auxiliary factor related to virus replication.

[0003] The mortality rate of PEDV-infected piglets is extremely high, and the incidence rate is higher in the cold season at the end of winter and the beginning of spring. Since the current vaccine against PED does not have high specificity, and the SIgA protection time provided by the prevention and control means mainly based on the feeding of pigs is short, PEDV continues to spread in the immune pig population.

[0004] Due to the structural characteristics of the single-stranded virus, it shows a high mutation tendency, and the S gene plays an important role in the genetic evolution of the virus, and the S gene variation is the main reason for the change of the virulence of the strain. At present, according to the sequence analysis of the S gene of PEDV strain, different strains of PEDV can be divided into two genotypes of G I and G II, and G I genotype can be divided into G Ia type and G Ib type. At present, the main epidemic strain in China is G II type, which has undergone great variation compared with the classic strain CV777 of PEDV, therefore, the existing vaccine is difficult to effectively prevent and control. Determining the genetic evolution characteristics of the epidemic strain and the pathogenicity of the strain is the key work for later vaccine research. In order to prepare a highly specific vaccine for preventing and controlling G II type porcine epidemic diarrhea, it is necessary to obtain the related virus strain, therefore, in order to prepare a vaccine for preventing and controlling G II type porcine epidemic diarrhea, it is necessary to isolate a G IIa type porcine epidemic diarrhea virus strain. SUMMARY

[0005] In order to obtain a G IIa type porcine epidemic diarrhea virus strain, the present application provides a G IIa type porcine epidemic diarrhea virus strain and application. The strain provided by the present application is a PEDV strong pathogenicity strain, which can cause vomiting and diarrhea in piglets after infection, so that the development of piglets is relatively slow, the hair is rough, severe dehydration, depression, and constant tremor. The inactivated vaccine prepared by using the strain has good immunogenicity. It can be used for preventing porcine epidemic diarrhea caused by PEDV.

[0006] The present application provides a porcine epidemic diarrhea virus (PEDV) strain, which is named LZ202401 and preserved in China Center for Type Culture Collection on October 15, 2024, with a preservation number of CCTCC NO:V202488.

[0007] The strain provided by the present application can cause vomiting and diarrhea in piglets after infection, so that the development of piglets is relatively slow, the hair is rough, severe dehydration, depression, and constant tremor, so it is a PEDV strong pathogenicity strain. The inactivated vaccine prepared by using the strain has good immunogenicity after immunizing pigs.

[0008] The present application further provides an application of the porcine epidemic diarrhea virus strain in preparing a product for preventing and / or treating diseases caused by porcine epidemic diarrhea virus.

[0009] Further, the product is a vaccine or a drug.

[0010] Further, the vaccine takes inactivated strain LZ202401 as the only effective component.

[0011] The application provides a vaccine composition, which comprises an effective amount of the porcine epidemic diarrhea virus strain LZ202401.

[0012] Further, the vaccine composition further comprises a vaccine adjuvant.

[0013] Further, the vaccine composition is composed of an inactivated vaccine stock solution and a Montanide ISA206 adjuvant, wherein the inactivated vaccine stock solution is obtained by culturing and enriching inactivation of the porcine epidemic diarrhea virus strain LZ202401.

[0014] Further, the virus titer in the inactivated vaccine stock solution is 10 6.5 TCID 50 / 100 μL.

[0015] The application further provides a preparation method of the vaccine composition, which comprises the following steps.

[0016] The porcine epidemic diarrhea virus strain LZ202401 is co-cultured with Vero cells, and when more than 80% of the Vero cells show typical cytopathic effects, the virus-infected cell suspension is collected by freezing and thawing 3-4 times, and the cell fragments are removed by centrifugation at 10,000-12,000 rpm for 20-25 min at 4 ℃, to obtain a virus stock solution;

[0017] The collected virus stock solution is slowly added to a sucrose cushion, and then centrifuged at 40,000-50,000 rpm for 3.5-4 h, and the precipitate is collected with sterile PBS, and the virus solution is enriched and diluted to obtain a high-titer virus solution, and then inactivated to obtain an inactivated vaccine stock solution;

[0018] The inactivated vaccine stock solution is uniformly mixed with the Montanide ISA206 adjuvant to obtain the vaccine composition.

[0019] Further, the first centrifugation condition is 12,000 rpm for 20 min.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] The application provides a porcine epidemic diarrhea virus strain LZ202401, which is a GIIa type PEDV strain, and the first day of 4-day-old piglets orally infected with the strain shows vomiting and diarrhea, and then the condition is aggravated, the piglets are more sluggish, the hair is rough, severe dehydration, the spirit is depressed, and the tremor is unceasing. It is shown that the strain is a strong pathogenicity strain of PEDV. The strain LZ202401 is prepared into a vaccine, and the vaccine is used for immunizing pigs, and it is shown that a high level of IgG antibody is produced in serum after immunization, and the SIgA level in colostrum of immunized sows is extremely significantly higher than that of the control group of sows. The 4-day-old piglets are subjected to a challenge protection test, and the survival rate of the immunized group of newborn piglets is 80% after challenge, and the control group of newborn piglets has no survival. The prepared porcine epidemic diarrhea virus inactivated vaccine has good protection. The prepared vaccine has good immunogenicity.

[0022] Biological material preservation information

[0023] LZ202401, referred to as a porcine epidemic diarrhea virus strain in the application, has been preserved in the China Center for Type Culture Collection on October 15, 2024, and the preservation number is CCTCC NO: V202488. The address of the preservation unit is Wuhan, Wuhan University, China, and the postcode is 430072. The classification and naming are porcine epidemic diarrhea virus LZ202401 Porcine epidemic diarrhea virus. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0025] Figure 1 The figure is the RT-PCR detection result of the biological material, in which, negative represents a negative control, positive represents a positive control, and sample represents a sample.

[0026] Figure 2 It is the pathological condition of Vero cells after infection and the RT-PCR detection virus passage result;

[0027] In the figure, A is the pathological change result of Vero cells after infection, Mock represents Vero cells without infection treatment, and LZ202401 represents Vero cells with infection treatment.

[0028] B is the RT-PCR detection result of each generation of cell culture from the first generation to the sixth generation.

[0029] Figure 3 are the results of indirect immunofluorescence test and Western blot verification of PEDV infection of Vero cells;

[0030] In the figure, A is the result of indirect immunofluorescence test of virus infection of Vero cells, Mock represents Vero cells without virus treatment, and LZ202401 represents Vero cells with virus treatment;

[0031] B is the result of Western blot verification.

[0032] Figure 4 is a transmission electron microscope image of LZ202401 virus particles.

[0033] Figure 5 is the result of virus purification of LZ202401 and the TCID 50 determination result of LZ202401 virus;

[0034] In the figure, A is the result of neutral red staining observation in LZ202401 strain plaque purification, Mock represents Vero cells without virus treatment, and LZ202401 represents Vero cells with virus treatment;

[0035] B is the TCID 50 determination value of LZ202401 strain.

[0036] Figure 6 is the sequence analysis of S gene of LZ202401 virus strain;

[0037] In the figure, A is the sequence homology analysis of S gene of LZ202401 strain;

[0038] B is the phylogenetic tree of S gene sequence of LZ202401 strain;

[0039] C is the comparison of mutation sites in COE region of S protein.

[0040] Figure 7 is the sequence homology analysis of whole genome sequence of LZ202401 virus strain.

[0041] Figure 8 is the diarrhea and vomiting incidence of experimental piglets after infection with LZ202401 strain;

[0042] In the figure, A is the feces condition of piglets;

[0043] B is the vomiting condition of piglets.

[0044] Figure 9 is the clinical diarrhea performance, diarrhea comprehensive score and survival curve of piglets after infection with LZ202401 strain;

[0045] A, the anus of piglets infected with LZ202401 strain for 24h and 48h;

[0046] B, the diarrhea score of piglets;

[0047] C, the survival curve of piglets.

[0048] Figure 10 A, the appearance of infected piglets and the abdominal cavity of piglets after autopsy;

[0049] A, the difference in body size of piglets in each group after infection;

[0050] B, the pathological changes in the abdominal cavity after autopsy (the lesion characteristics are marked by red arrows).

[0051] Figure 11 The viral content in each segment of the intestinal tissue of each treatment group after infection.

[0052] Figure 12 The observation of piglet intestinal tissue sections (10x).

[0053] Figure 13 The antibody level of inactivated vaccine in serum and colostrum;

[0054] A, the IgG titer in serum of the control group and the immunized group before and after immunization;

[0055] B, the SIgA titer in the postpartum colostrum of the control group and the immunized group.

[0056] Figure 14 The survival curve of piglets after challenge. DETAILED DESCRIPTION

[0057] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application. The experimental methods described in each embodiment of the present application are conventional methods, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0058] Example 1: Isolation, identification, purification and whole genome sequencing analysis of a GIIa type porcine epidemic diarrhea virus strain.

[0059] I. Treatment of pathological material and design and synthesis of PCR primers

[0060] The small intestine tissue was taken from a pig farm in Gansu province after the pig was dissected, and was grinded with sterile normal saline according to the ratio of 1:8, and then was repeatedly frozen and thawed for 3 times, and was centrifuged at 4℃, 12000 rpm for 20 min, and the supernatant was taken, and the supernatant was filtered through a 0.22 μm filter screen, and then the nucleic acid was extracted from the supernatant. The detection primers of PEDV, TGEV, PDCoV, PoRV, PCV2, PRV and PPV were designed, and were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., and the primer sequence information was shown in Table 1 (F represents the upstream primer sequence; R represents the downstream primer sequence).

[0061] Table 1 primer information

[0062]

[0063] II. Detection of pathogens

[0064] The extracted nucleic acid was used as a template to detect the related pathogens by the detection primers of PEDV, TGEV, PDCoV, PoRV, PCV2, PRV and PPV, the nucleic acid extracted from the cells without infection was used as a negative control, and the nucleic acid extracted from the cells with infection was used as a positive control. The reaction system was 20 μL: 2x M5 Hiper plus Taq Hifi PCR Mix 10 μL, 0.5 μL of each upstream and downstream primer, 1 μL of DNA / cDNA, and 8 μL of ddH2O; the PCR amplification program was 95℃ pre-denaturation for 5 min; 94℃ denaturation for 25 s; annealing for 25 s according to the annealing temperature of each primer; 72℃ extension for 20 s, 35 cycles; 72℃ final extension for 10 min, and then 4℃ storage. The amplification products were detected by 1% agarose gel electrophoresis under the condition of 130 V, 15 min.

[0065] The genomic nucleic acid (extracted nucleic acid) of the sample was amplified by PCR, and the amplification products were used for pathogen detection by 1% agarose gel electrophoresis, and the results were shown in Figure 1 As shown in the figure, one sample successfully amplified a target fragment with a size of about 273 bp, and combined with the size of the target fragment when the primer was designed, it was speculated to be a PEDV positive product, and the identification results of other amplification products were negative.

[0066] III. Virus isolation

[0067] Vero E6 cells were subcultured into T25 cell culture flasks, and when the cell confluence reached more than 90%, the culture medium was discarded and the cells were washed with sterile PBS three times. Then, 1 mL of the supernatant of the sample was mixed with 4 mL of serum-free DMEM medium (purchased from Promocell) and inoculated into Vero cells. Subsequently, 10 μg / mL of trypsin was added, and the cells were cultured in a cell incubator at 37°C and 5% CO2. The cell state was observed daily, and when the cells showed obvious pathological changes, the culture was harvested. After repeated freezing and thawing at -80°C for three times, the virus-infected cell suspension was collected and centrifuged at 4°C and 12,000 rpm for 20 min to remove cell debris. The virus stock solution was obtained, filtered through a 0.22 μm filter to remove bacteria, and stored at -80°C. The virus was transmitted to the 6th generation using the above inoculation method, and the nucleic acid of the cell culture of each generation was extracted and used as a template for PCR amplification and agarose gel electrophoresis to detect PEDV.

[0068] The serum-free DMEM medium formula is as follows: 4500 mg / L D-glucose, 3700 mg / L NaHCO3, 584 mg / L L-glutamine, 110 mg / L sodium pyruvate, and 15 mg / L phenol red are dissolved in 500 mL of high-purity triple distilled water. According to a volume of 500 mL, 100 U / mL penicillin and 0.1 mg / mL streptomycin are added, and the pH is adjusted to 7.2 using NaOH.

[0069] The results are shown in Figure 2 A, the supernatant of the sample was inoculated into Vero cells, and after 48 h of blind transmission to the 2nd generation, obvious cell pathological changes were observed. The cell outline disappeared, the cell surface was full of granules, a large number of cells were shed, and the cells were connected by a network. The untreated Vero cells were oval, grew in a single layer, arranged in an orderly manner, and rarely shed. The results showed that the strain could produce significant pathological changes on Vero cells. The virus was stably transmitted to the 6th generation, and the virus liquid of each generation was collected and filtered through a 0.22 μm filter to remove bacteria, and stored at -80°C. Through detection of the cell culture of each generation, a specific band with a product size of 273 bp was amplified (B), which was consistent with the expected result. Figure 2

[0070] Four, identification of the virus

[0071] ​Indirect immunofluorescence: the 6th generation of virus liquid was inoculated in Vero cells for 24 hours, the culture medium was discarded, and the cells were washed with sterile PBS for 3 times, 4% paraformaldehyde was added for fixation for 25 min; the cells were washed with sterile PBS for 3 times, 0.1% Triton X-100 was added for 10 min at room temperature; the cells were washed with sterile PBS for 3 times, and the blocking solution was added for blocking for 1 hour; the rabbit-derived PEDV N protein polyclonal antibody (1:100) was incubated at room temperature for 1 hour; the cells were washed with sterile PBS for 3 times, and the fluorescent secondary antibody (1:500) was added for incubation for 1.5 hours in the dark; the cells were washed with sterile PBS for 3 times, and then DAPI was added for incubation for 10 min in the dark; after washing with sterile PBS, the cells were observed under a fluorescence inverted microscope.

[0072] Western blotting: the PEDV classical strain CV777 and the 6th generation of virus liquid of the isolated strain were inoculated in Vero cells, respectively, the culture medium was discarded after 48 hours of virus infection, the cells were washed with PBS for 3 times, the cells were collected into a 1.5 mL centrifuge tube by using a cell scraper, the cells were lysed by adding RIPA lysis buffer, PMSF was added at a ratio of 1:100 of protease inhibitor (PMSF) to RIPA lysis buffer, and the cells were slowly lysed in an ice box on a slow shaker for 30 min, centrifuged at 12000 rpm for 10 min, the supernatant was aspirated, and the protein was denatured by adding protein loading buffer and boiling in boiling water for 10 min, and then the protein was spotted. The protein was transferred to a PVDF membrane under the conditions of electrophoresis at 90 V for 30 min and 120 V for 60 min, and membrane transfer at 220 mA for 60 min, the membrane was blocked for 2 hours after blocking with blocking solution; the rabbit-derived PEDV N protein polyclonal antibody (1:1000) was incubated on a shaker at 4°C overnight; the secondary antibody (1:10000) was incubated at room temperature for 2 hours after PBST washing; the membrane was immersed in the luminescence solution for automatic exposure after PBST washing.

[0073] Transmission electron microscopy: the 6th generation of virus liquid of the isolated strain was inoculated into monolayer dense Vero cells, and when the cytopathic effect reached more than 80%, the virus liquid was harvested, centrifuged at 12000 rpm for 10 min at 4°C, the supernatant was aspirated, centrifuged at 30000 rpm for 2 hours at 4°C, resuspended with an appropriate amount of sterile PBS until the precipitate was completely dissolved, and then an appropriate amount of the obtained suspension was added dropwise to an ultrathin carbon film copper grid for adsorption for 5 min, washed with PBS, and then an appropriate amount of tungsten-phosphoric acid negative stain was added dropwise, dried, and then observed under a microscope.

[0074] Indirect immunofluorescence, Western blotting, and transmission electron microscopy identification results: the 6th generation of virus liquid of the isolated strain was inoculated into monolayer dense Vero cells, and the uninfected group was used as a control, and the cells were fixed 24 hours after inoculation. The results of IFA identification are shown in Figure 3 Figure 3 ​A), the control group has no green fluorescence. The isolated strain is inoculated into Vero cells, and Western Blotting is used for verification. The results show that, compared with CV777, the isolated strain shows a target protein at 57 kDa (Fig. 3B). The virus supernatant is subjected to ultracentrifugation at 4°C, and is negatively stained with tungsten phosphate. Under electron microscopy, a field of view can be observed to have spherical particles with a diameter of 95 nm to 190 nm, showing the typical morphological characteristics of a coronavirus, and the surface can be seen to have a viral envelope and filopodia Figure 4 No other morphological virus particles are observed, further indicating that the isolated strain is a coronavirus PEDV, which is named as LZ202401 strain.

[0075] V. Virus plaque purification and determination of virus titer

[0076] Plaque purification: the virus liquid of the 6th generation is diluted with cell growth maintenance liquid and treated with 8 gradients (10 -2 ~ 10 -9 ) per hole. After adsorption for 2 hours, the virus liquid is discarded and slowly washed with sterile PBS. Low-melting-point agarose and preheated serum-free 2x DMEM medium are mixed in equal volumes, and 2 mL per hole is added. After cooling and solidification at room temperature, it is inverted and cultured in a cell culture incubator at 37°C and 5% CO2. Untreated Vero cells are used as a control. The cytopathic effect is observed every day, and 1 mL of the above mixture (containing 0.002% neutral red) is added to the cells twice after 48 hours. After 12 hours of inverted culture, appropriate size plaques are picked for identification and enrichment culture.

[0077] Determination of virus titer: the purified virus liquid is inoculated into cells, and the virus supernatant of 12h, 24h, 36h, 48h, 60h, and 72h is collected. The dilution is sequentially diluted according to 1x10 -1 ~ 1x10 -8 , and 100 μL of each is inoculated into pre-prepared 6 96-well plates of Vero cells. After adsorption for 2 hours, 100 μL of cell maintenance liquid is added to each hole. Untreated Vero cells are used as a control. It is placed in a culture incubator and cultured. After 72 hours, the number of cytopathic holes is observed and recorded, and the TCID 50 of the virus is calculated by the Reed-Muench method.

[0078] The results are shown in the figure. The isolated strain is diluted by a factor of 2 and inoculated into 6-hole plates of Vero cells, and the untreated group is used as a control. After 48 hours of inoculation, obvious cytopathic effect is observed in the cells, and neutral red staining shows that the inoculated group has a plaque with a diameter of about 2.8 mm Figure 5A), while the control group had no plaque formation, and the subsequent culture and identification of the plaque were carried out by picking the plaque of the virus group. The virus content after purification was determined, and the virus titer was calculated by the Reed-Muench method as 10 6.0 TCID 50 / mL( Figure 5 of B).

[0079] Six, genomic sequence analysis

[0080] The purified virus liquid was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. By comparing the whole genome sequence of each genotype of PEDV classical strain at home and abroad, the sequencing results were analyzed and spliced by using DNA MAN software. The S gene sequences and whole genome sequences of typical PEDV strains and PEDV strains prevalent in different regions and at different times in recent years were obtained from the GenBank database, and the nucleotide homology of the strains was analyzed by using DNAstar software. The S protein antigen epitope COE region mutation site of the isolated strain and the representative strains of different genotypes of PEDV was compared and analyzed. The S gene phylogenetic tree was drawn by using MEGA software.

[0081] The sequencing results were processed and spliced by using DNAMAN software, and the full length of LZ202401 S gene was 4161 bp. By comparing the S gene sequences of LZ202401 and 45 representative PEDV strains at home and abroad, it was found that LZ202401 had the highest homology with HK2021 (GenBank accession number: OL762457.1) and SXSL (GenBank accession number: MZ241108.1), which was 99.4%, and had higher homology with CH / JXXG / 11 / 2020 (GenBank accession number: MZ161016.1), which was 99.3%; The homology with GⅠ, GⅡa type vaccine strains CV777 (GenBank accession number: AF353511.1), AH2012 (GenBank accession number: KC210145.1) was 93.8%, 98.1% respectively; The homology with GⅡb type classical strain AJ1102 (GenBank accession number: JX188454.1) was 97.3%( Figure 6 A). LZ202401 and HK2021, SXSL belong to GⅡa type( Figure 6 B); and the Japanese strain, Korean strain and American strain have distant genetic relationship. By analyzing the mutation sites of the S protein antigen epitope COE region, it was found that LZ202401 had 12 mutation sites with different subgroups of representative strains( Figure 6 C).

[0082] Whole genome sequence analysis results: the whole genome of LZ202401 is 28038 bp. By comparing the whole genome sequence of LZ202401 with that of 45 representative PEDV strains at home and abroad, it is found that LZ202401 has the highest homology with SXSL, which is 99.6%; it has a higher homology with HK2021, which is 99.5%; and it has a lower homology with Asian and European strains. Figure 7 ).

[0083] The above results show that the virus successfully isolated from sick pigs in pig farms in Gansu Province is a porcine epidemic diarrhea virus. The strain is named LZ202401. The strain is a GⅡa type PEDV strain. It is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO:V202488, the preservation date October 15, 2024, and the preservation address Wuhan, Wuhan University, China.

[0084] Example 2: Animal regression test of porcine epidemic diarrhea virus LZ202401.

[0085] I. Purchase of piglets and grouping of piglet test

[0086] Twelve 4-day-old healthy piglets produced by the same pregnant sow were randomly divided into four groups according to every three piglets in one group, which were control group, low-dose, medium-dose and high-dose infection group, and the piglets in each treatment group were fed in different rooms.

[0087] The 12 4-day-old healthy piglets for animal test were purchased from a pig farm in Shaanxi Province, and the pregnant sow was not inoculated with viral diarrhea vaccine before delivery. The piglets had normal feces and appetite.

[0088] II. Piglet infection test

[0089] After the piglets adapted to the environment, the control group piglets were fed with 2 mL of serum-free DMEM medium, and the infected group piglets were fed with the virus liquid enriched after plaque purification in Example 1 above. The low-dose, medium-dose and high-dose infection group piglets were fed with 1 mL, 1.5 mL and 2 mL of virus liquid (virus titer was 10 6.0 TCID 50 / mL) respectively, and the low-dose and medium-dose infection group piglets were supplemented with serum-free DMEM medium to 2 mL.

[0090] From the day of infection, the body weight, vomiting condition and feces consistency of the piglets were measured and recorded every day.

[0091] Figure 8-9 The results are shown in the table below. Figure 8 As shown in the table, the piglets in the test group appeared vomiting and diarrhea after 1 day of oral inoculation of LZ202401 strain, and the piglets in the high-dose infection group died on the second day after infection, and the piglets in the medium and low-dose infection groups discharged liquid, yellow fecesFigure 8 (A) Diarrhea was more pronounced in piglets on the 3rd and 4th days after infection, with watery, pale yellow feces and frequent vomiting. Figure 8 In group B), deaths occurred successively in the medium and low dose infection groups. Clinical observation of the piglets' anuses during the experiment revealed varying degrees of perianal redness and swelling, rectal prolapse, and persistent diarrhea in all groups. Figure 9 (A). A comprehensive score was given to the clinical manifestations of diarrhea in each group of piglets. Figure 9 (B) By recording mortality in low, medium, and high-dose infection groups, survival curves for piglets were plotted. Figure 9 (C). During the infection period, the control group piglets had a normal diet and developed well, while the experimental group piglets showed slower development, rough coats, severe dehydration, depression, and persistent tremors.

[0092] III. Piglet necropsy and tissue sampling

[0093] Five days after oral infection of piglets, surviving piglets in each treatment group were euthanized and then eviscerated. The condition of the piglets' abdominal cavity was observed, and intestinal tissue was collected from each group to detect the viral content. At the same time, the intestinal tissue was fixed in 4% paraformaldehyde solution for the preparation of tissue sections.

[0094] Piglets in both the control and experimental groups were euthanized and then necropsy was performed. Figure 10 (A). Autopsy revealed undigested curd in the stomachs of piglets in the experimental group; thinning and congestion of the small intestinal wall; edema, distension, and filling with yellow watery contents in all segments of the small intestine; and varying degrees of bloating and edema in all segments of the large intestine. In the low and medium dose infection groups, the small intestines of piglets showed severe edema, appearing transparent, and the intestinal walls were thinned; in the high dose infection group, the intestinal tissues were severely congested, containing large amounts of yellow contents, and the mesentery showed dendritic congestion. Figure 10 (B).

[0095] IV. Analysis of Viral Content in Intestinal Tissue

[0096] Virus content analysis of intestinal tissue: After necropsy, different intestinal tissues of piglets from each group were taken, and nucleic acid was extracted according to the tissue processing method in Example 1 above. The virus content in each tissue was then detected.

[0097] Different viral loads were detected in different segments of the small and large intestines, with the highest viral loads detected in the jejunum and ileum, and relatively lower viral loads detected in the duodenum. A certain amount of virus was detected in all segments of the large intestine, but the viral load was lower compared to that in the small intestine. The viral loads in different tissues of the intestinal segments of piglets in the low, medium, and high dose infection groups showed the same trend. Figure 11 ).

[0098] V. Intestinal Histopathology

[0099] The tissue is taken out from the fixing liquid, trimmed into cross-section tissue blocks with a thickness of about 0.3mm, and placed in an embedding box, and washed by flowing tap water overnight; the tissue is dehydrated by using an automatic dehydration machine, and after being transparentized by xylene, the tissue is immersed in wax. Hot wax is injected into a metal frame by using an embedding machine for embedding; after the wax block is completely solidified, the metal frame is removed, and the wax block is fixed on a microtome for slicing, and the slices with a thickness of 5um are laid flat in warm water with the front surface upward, and then taken out by using a glass slide, and after being marked, the slices are dried, placed on a staining rack, and subjected to HE staining. An equal volume of neutral resin and xylene is mixed and used for sealing, and after being dried, the sealed slices are observed under a microscope.

[0100] Histopathological observation: compared with the control piglets, the villus length of the duodenum of the test piglets is shortened, shed, reduced in number, and the cells are necrotic; the villus of the jejunum is largely shed, atrophied, shortened, and the cells are degenerated and necrotic; the villus length of the ileum is shortened, the spacing is widened, and the lymphocytes are infiltrated; the villus of the cecum and the ileum is broken; and the mucosal muscle layer and the connective tissue of each segment of the large and small intestines are all congested. Figure 12

[0101] The above results show that the virus successfully isolated from the small intestinal tissue of sick pigs in a pig farm in Gansu Province is a PEDV highly pathogenic strain.

[0102] Example 3: Preparation method of porcine epidemic diarrhea virus inactivated vaccine

[0103] I. Virus propagation

[0104] The Vero cells are subcultured into a T25 cell culture bottle, and when the cell confluence degree is 90%-95%, the culture medium is discarded and washed with sterile PBS for 3 times, and the virus liquid enriched after the plaque purification in the above example 1 (virus titer is 10 6.0 TCID 50 / mL) is added in a volume of 500ul, and after the virus is adsorbed to the cells for 2h, the virus liquid is discarded, and washed with sterile PBS for 3 times, and then the virus is propagated by adding serum-free DMEM maintenance liquid;

[0105] II. Virus harvesting

[0106] After the virus is continuously cultured, when 80% of the Vero cells show typical cytopathic effect, the cells are frozen and thawed 3 times in a-80℃ refrigerator, the virus-infected cell suspension is collected, centrifuged at 4℃ and 12000rpm for 20min, and the cell debris is removed to obtain the virus stock solution;

[0107] III. Determination of virus titer

[0108] ​The collected virus stock solution was slowly injected into a 20% sucrose cushion, and the virus was enriched by low-speed ultracentrifugation at 4°C and 45,000 rpm for 4 h. The precipitate was collected with sterile 1xPBS, and the enriched virus solution was diluted with serum-free DMEM medium to a virus titer of 10 6.5 TCID 50 / 100 μL to obtain a high-titer virus solution, which was stored at -80°C for standby use.

[0109] Four, virus inactivation treatment

[0110] The high-titer virus solution was thawed at room temperature, and a formaldehyde solution with a final concentration of 0.1% was added. After uniform mixing, the solution was inactivated at 37°C and 200 rpm for 48 h on a shaking table. Subsequently, sodium sulfite was added to neutralize the formaldehyde solution, and an inactivated vaccine stock solution of PEDV was obtained, which was stored in a refrigerator at 4°C for standby use.

[0111] Five, vaccine preparation

[0112] The inactivated vaccine stock solution was mixed with Montanide ISA206 adjuvant according to the instructions at an equal volume to obtain a directly usable inactivated vaccine for porcine epidemic diarrhea virus.

[0113] Example 4 Safety test of inactivated vaccine for porcine epidemic diarrhea virus

[0114] One, inactivation effect test of vaccine

[0115] The prepared vaccine was inoculated into Vero cells according to the virus propagation method of Example 3 above, and a control group was set up. Continuous observation was carried out for one week. If cell pathological effects appeared during this period, it indicated that the virus was not completely inactivated. If no cell pathological effects were observed, the same method was used for five generations. If cell pathological effects appeared during the generation, it indicated that the virus was still not completely inactivated. If no cell pathological effects appeared after five generations, it indicated that the virus was completely inactivated. The inactivated vaccine of the present application was completely inactivated after the inactivation effect test.

[0116] Two, physical property test of vaccine

[0117] Two mL of vaccine was centrifuged at 3,000 rpm for 15 min, and whether stratification occurred was observed. The vaccine of the present application did not show stratification.

[0118] Three, sterility test of vaccine

[0119] The test was carried out according to the appendix of Chinese Veterinary Pharmacopoeia (2020 edition), and no bacterial growth was observed.

[0120] Four, inoculation safety test of vaccine

[0121] Select 12 healthy pregnant sows which are not inoculated with viral diarrhea vaccine, and randomly divide them into 4 groups according to every 3 sows as a group, which are control group and low, medium and high dose immunization groups. The immunization groups are inoculated with the vaccine according to 2 mL, 4 mL and 6 mL inoculation amount respectively, and the control group is inoculated with the same amount of sterile normal saline. They are fed under the same condition, and continuously observed for one week. The test shows that the pregnant sows in the control group and the immunization groups have normal feeding, good mental state, and no death occurs.

[0122] The above test shows that the inactivated vaccine has good safety.

[0123] Example 5: Preliminary evaluation of the effect of inactivated vaccine

[0124] I. Determination of immunization effect of inactivated vaccine

[0125] 6 healthy pregnant sows are randomly divided into a control group and an immunization group, and the serum is collected before immunization. The two groups of sows are immunized once at 28 days before delivery and 14 days before delivery respectively. The control group is injected with 4 mL of sterile normal saline per sow, and the immunization group is injected with 4 mL of inactivated vaccine per sow. The colostrum and serum of the sows are collected after delivery, and the neutralizing antibody titer is measured. The IgG in the serum of the sows after immunization and the SIgA in the colostrum of the sows after delivery are detected respectively. The immunization group shows that a higher level of IgG antibody is produced in the serum after immunization (A), and the SIgA level in the colostrum of the sows after delivery is extremely significantly higher than that of the control group sows (B). Figure 13 Figure 13

[0126] II. Challenge protection test of piglets

[0127] After the sows are delivered, 6 4-day-old piglets are randomly taken back from the control group and the immunization group for challenge protection test. The piglets in the two groups are fed in different rooms. After the piglets adapt to the new environment, each piglet is orally challenged with 2 mL of virus. The diarrhea and death time of the piglets in the control group and the immunization group are recorded. The survival rate of the newborn piglets in the immunization group after challenge is 80%, and there is no survival of the newborn piglets in the control group ( Figure 14 ).

[0128] The protection effect of PEDV vaccine is mainly determined by the level of secretory immunoglobulin (SIgA) in the colostrum of sows. The above results show that the inactivated vaccine for porcine epidemic diarrhea virus prepared by the present application has good protection.

[0129] ​​It can be seen from the above examples that starting from the intestinal segment tissue of the piglets with clinical diarrhea, a strain of PEDV which can produce significant lesions on Vero cells and can be stably passaged is obtained through virus extraction, isolation, identification and purification, and the sequencing and genetic evolution analysis show that the strain belongs to GIIa genotype. Animal experiments show that LZ202401 has strong pathogenic characteristics of PEDV, mainly damaging the digestive system of piglets. The inactivated vaccine prepared for the strain has good immunogenicity. The application provides a theoretical basis for understanding the epidemic situation, molecular characteristics and genetic variation characteristics of the current PEDV variant strain, and provides technical support for the prevention and control of PEDV, and has a wide application prospect.

[0130] Although preferred embodiments of the application have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once they have been given the basic inventive concept.

[0131] Obviously, those skilled in the art can make various modifications and changes to the application without departing from the spirit and scope of the application. Thus, if these modifications and changes of the application belong to the scope of the claims of the application and equivalent technologies thereof, the application also intends to include these modifications and changes.

Claims

1. A vaccine composition, characterized in that, The vaccine composition is composed of an inactivated vaccine stock of porcine epidemic diarrhea virus strain LZ202401 and Montanide ISA206 adjuvant, the inactivated vaccine stock is obtained after enrichment and inactivation of porcine epidemic diarrhea virus strain LZ202401 culture; the porcine epidemic diarrhea virus strain LZ202401 was deposited in the China Center for Type Culture Collection on October 15, 2024, and the deposit number is CCTCC NO: V202488.

2. The vaccine composition of claim 1, wherein, The virus titer in the inactivated vaccine stock is 10 6.5 TCID 50 / 100 μL.

3. A method of preparing a vaccine composition according to any one of claims 1 to 2, characterized in that, The method comprises the following steps: The porcine epidemic diarrhea virus strain LZ202401 is co-cultured with Vero cells, when more than 80% of the Vero cells show typical cytopathic effect, the virus infected cell suspension is collected, the cell debris is removed by centrifugation at 10000 rpm-12000 rpm for the first time for 20 min-25 min, and the virus stock is obtained; The collected virus stock is slowly added to the sucrose cushion, and the second centrifugation is performed at 40000 rpm-50000 rpm for 3.5 h-4 h, the precipitate is collected with sterile PBS, and the virus liquid is enriched and diluted to obtain a high-titer virus liquid, and the inactivated vaccine stock is obtained after inactivation; The inactivated vaccine stock is mixed with Montanide ISA206 adjuvant to obtain the vaccine composition.

4. The method of preparing a vaccine composition according to claim 3, wherein, The first centrifugation condition is 12000 rpm for 20 min.

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