PEDV (Porcine Epidemic Diarrhea Virus) strain and preparation method and application of gastric acid-resistant yolk antibody

Abstract

The invention discloses a preparation method and application of a GIIc type PEDV (Porcine Epidemic Diarrhea Virus) strain and a gastric acid resistant specific egg yolk antibody thereof, the PEDV strain is a GIIc type clinical predominant prevalent strain, and has good cell adaptability after plaque purification and domestication; the strain can be used for developing PEDV attenuated live vaccines, inactivated vaccines, novel genetic engineering vaccines, preventive and therapeutic antibodies and diagnostic reagents, and solves the technical problems that the existing vaccines are insufficient in protective power and the GIIc type PEDV clinical isolated strain is low in in-vitro culture titer; meanwhile, the invention discloses a core-shell nanoparticle antibody coating technology based on zein / carboxymethyl chitosan (Zein / CMCS), the PEDV egg yolk antibody IgY can be protected from being damaged by a strong acid environment and pepsase in the stomach of a pig, viruses in the intestinal tract are fully released and neutralized in the intestinal tract, PEDV infection is effectively prevented and treated, and the effect of preventing and treating porcine epidemic diarrhea is achieved. The problem that the neutralizing activity of the existing PEDV egg yolk antibody is sharply reduced after intragastric administration is solved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for preparing and applying a specific egg yolk antibody against GⅡc type PEDV virus strain and its resistance to gastric acid. Background Technology

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV). Typical symptoms include watery diarrhea, dehydration, and vomiting. The disease is characterized by high infection rates, high morbidity, and high mortality rates, and is particularly harmful to suckling piglets, especially those under 7 days old, where morbidity and mortality can approach 100%. Since 2010, PEDV variants with even higher infection and mortality rates, stronger pathogenicity, and greater immune evasion capabilities have emerged, causing the deaths of tens of millions of piglets worldwide and resulting in enormous economic losses to the pig industry in my country and globally.

[0003] PEDV belongs to the genus α-coronavirus (α-CoV) within the family Coronaviridae, subfamily Coronaviridae. It is an enveloped, single-stranded, positive-sense RNA virus with a diameter of 95–190 nm. The viral genome is approximately 28 kb in length. Based on the genetic diversity of the S gene, PEDV can be divided into two main genotypes: GⅠ (classical strain) and GⅡ (variant strain). GⅠ is further divided into two subtypes, GⅠa and GⅠb, with CV777 and DR113 as representative strains, belonging to low pathogenicity. GⅡ is a highly pathogenic variant strain, highly pathogenic to suckling piglets, and is further divided into GⅡa, GⅡb, and GⅡc. Animal experiments show that the GⅡc strain has a higher mortality rate in piglets, with clinical symptoms such as diarrhea and vomiting appearing as early as 12 hours after infection. Existing commercial vaccines are prepared from GⅠb, GⅡa, and GⅡb strains, and cannot provide effective protection against the circulating GⅡc strain; immunized piglets still experience severe diarrhea and death. Developing inactivated vaccines, live attenuated vaccines, novel vaccines (such as mRNA vaccines and virus-like particle vaccines), and therapeutic antibodies (including egg yolk antibodies) against prevalent GIIc strains has become an urgent need to control the global epidemic and reduce piglet mortality.

[0004] Immunoglobulin Y (IgY) is an immunoglobulin extracted from the yolks of immunized hens, functionally similar to mammalian IgG. In recent years, it has shown broad application prospects in the prevention and treatment of animal diseases. High levels of specific IgY antibodies present in the blood of immunized laying hens accumulate in the yolk via a receptor-mediated active transport mechanism. Compared to mammalian antibody production, yolk antibody production does not require euthanasia to collect blood and separate serum; it allows for direct and continuous collection and purification of highly immunized eggs. The purification technology is simple and has significant advantages such as low cost and high yield. Yolk antibodies not only provide passive immunity to chicks but also offer a feasible pathway for large-scale, non-invasive production of antigen-specific antibodies. Due to its unique advantages, it is widely used in the prevention and treatment of bacterial and viral diseases. Furthermore, given the global concern about antibiotic resistance and drug residues in animal products, IgY has been extensively studied as an antibiotic alternative.

[0005] Studies have shown that purified anti-dengue IgY extracted from goose eggs can neutralize dengue virus in vitro. When mice were infected with a lethal dose for 24 hours and then treated with anti-dengue IgY, the cure rate reached 100%. Orally administered specific IgY has good preventive and therapeutic effects against gastrointestinal pathogens. For example, IgY targeting the fimbrial antigen of porcine enterotoxin-producing *Escherichia coli* showed significant preventive effects in field trials, achieving a cure rate of 92% in piglets with diarrhea. IgY also shows potential in combating rotavirus diarrhea in humans and newborn animals, as it can specifically neutralize rotavirus particles in the intestine, showing promise as an important therapeutic strategy. Other studies have explored the in vitro inhibitory effects of IgY on foodborne zoonotic pathogens such as *Campylobacter jejuni*, *Salmonella*, and *Escherichia coli*, confirming that hyperimmune egg yolk IgY powder can reduce the viral load of *Campylobacter jejuni* in the cecum of broilers. Necrotic enteritis caused by Clostridium perfringens results in significant economic losses for the poultry industry. Hyperimmune IgY targeting this bacterium's antigens has demonstrated immunoprotective and therapeutic effects in broilers, which is particularly important given the current restrictions on antibiotic use in poultry farming. IgY prepared using inactivated goose astrovirus (GAstV) as an immunogen can be used to prevent and treat goose astrovirus infection, a serious threat to the goose industry. High-titer IgY has also been successfully prepared by oral immunization of hens against Riemerella anatipestifer, which causes respiratory diseases in chickens. While IgY has a wider pH stability range and higher tolerance to proteases compared to mammalian IgG, the highly acidic environment (pH≈2.0–2.5) and pepsin in the piglets' stomachs can irreversibly destroy egg yolk antibodies when administered orally for the prevention and treatment of intestinal infections, significantly impacting their preventative and therapeutic efficacy. Studies have shown that unprotected IgY antibodies are inactivated by over 90% after 30 minutes in simulated gastric fluid at pH ≤ 3.0. Therefore, there is an urgent need to develop a specific egg yolk antibody against the GIIc type PEDV virus strain and its resistance to gastric acid for the prevention and treatment of PEDV infection. Summary of the Invention

[0006] This invention addresses the technical problems of low viral titers in in vitro culture of clinical isolates of PEDV type GⅡc and poor immunoprotective efficacy of existing commercially available inactivated and attenuated PEDV vaccines. It also solves the technical problems of poor efficacy of existing PEDV egg yolk antibodies for the prevention and treatment of PEDV infection in piglets via oral administration, their susceptibility to gastric acid destruction, and low antibody utilization. This invention isolates the dominant circulating PEDV strain GⅡc HLJ04, and through multiple rounds of double-layer plaque purification, finally domesticates and screens to obtain the cell-adaptable PEDV HLJ04 P10 / PFU strain. This strain can be used for the research and development of PEDV inactivated vaccines, live vaccines, diagnostic reagents, and therapeutic antibodies. Furthermore, this invention develops a gastric acid-resistant PEDV IgY-specific egg yolk antibody using zein / carboxymethyl chitosan (Zein / CMCS) core-shell nanoparticle coating technology. This antibody resists the highly acidic environment of the pig stomach and the destruction by pepsin. After full release in the intestine, it exhibits good preventive and therapeutic effects against PEDV infection, overcoming the shortcomings of existing egg yolk antibodies.

[0007] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: This invention provides a strain of PEDV virus of type GⅡc, wherein the strain is HLJ04 P10 / PFU strain of type GⅡc, and the microbial preservation number is CTCC NO:V202576.

[0008] This invention also provides a method for preparing a GIIc type PEDV virus strain, comprising the following steps: (1) GⅡc type PEDV positive tissue samples were ground, frozen and thawed, and the supernatant was filtered and sterilized before being inoculated into cells for virus isolation. After inoculation, the samples were replaced with PEDV DMEM cell maintenance medium and continuously blindly passaged until Vero E6 cells showed typical lesions such as rounding, aggregation, and syncytia. The S gene of the isolates was sequenced and genetic evolution was analyzed.

[0009] (2) In order to improve the cell adaptability of PEDV clinical isolates and increase the viral titer, continuous purification and passage were carried out through double plaques. After different dilutions of HLJ04 virus solution were inoculated into Vero E6 cells, the first layer of covering material was added. After further culture, the second layer of covering material was added. The morphology of viral plaques was observed, and viral plaques were selected for inoculation into Vero E6 cells for cell passage.

[0010] Preferably, the isolates are identified by indirect immunofluorescence (IFA) and transmission electron microscopy after phosphotungstic acid negative staining.

[0011] Preferably, the PEDV DMEM cell maintenance medium contains a final concentration of 0.3 μg / mL trypsin, 0.3% trypsin-phosphate broth, and 0.02% yeast extract.

[0012] Preferably, the first layer of plaque purification coating is PEDV DMEM cell maintenance medium containing 0.6% low melting point agarose, and the second layer coating is formulated as 7.5% NaHCO3 (340 μL) + 0.33% neutral red (240 μL) + 1.2% low melting point agarose (10 mL) + 2×PEDV DMEM cell maintenance medium (10 mL).

[0013] Preferably, during plaque purification, a second layer of coating is added 48–72 h after the first layer of coating is added, and the plaque is observed and picked after incubation for another 12–24 h after the second layer of coating is added.

[0014] Preferably, during plaque purification, large plaques are selected, inoculated into Vero E6 cells for acclimatization and passage, and plaque purification is carried out for at least 10 passages, with the viral titer after purification not lower than 10. 8.0 TCID 50 / mL.

[0015] This invention also provides the application of the above-mentioned strain in the development of inactivated PEDV vaccines, live attenuated vaccines, and PED-related diagnostic reagents for pigs.

[0016] This invention also provides a method for preparing a gastric acid-resistant specific egg yolk antibody against GⅡc type PEDV, comprising the following steps: (1) Vaccine preparation and immunization of laying hens: The PEDV HLJ04 P10 / PFU cell-adapted strain purified by plaque was inoculated into Vero E6 cells to prepare virus solution. After inactivation, water-in-oil type PEDV inactivated vaccine was prepared to immunize healthy laying hens during the laying period. After the first immunization, three booster immunizations were carried out every 21 days. High-immunity eggs were collected 14 to 35 days after the last immunization.

[0017] (2) Purification of egg yolk antibodies: After disinfection by soaking in 0.1% benzalkonium chloride, the eggs were crushed and the yolks were collected. Sterile deionized water was added at 7 times the volume of the yolks and the mixture was stirred and centrifuged to obtain the supernatant as the egg yolk aqueous extract. The egg yolk aqueous extract was acidified with dilute hydrochloric acid, and then caprylic acid was added while stirring to purify IgY. The pH of the purified IgY supernatant was adjusted back to 7.0, and ammonium sulfate solution was added for precipitation. The IgY antibody precipitate was dissolved in sterile PBS, placed in a dialysis bag and dialyzed overnight to remove salt and small molecule impurities. The dialyzed IgY solution was concentrated by ultrafiltration using a membrane with a molecular weight cutoff of 30 KD to obtain the PEDVIgY antibody solution.

[0018] (3) Acid-resistant coating of antibody: Prepare zein organic phase as core solution and carboxymethyl chitosan (CMCS) aqueous phase, adjust pH to 5.0-5.5 as shell solution; while stirring CMCS shell solution at high speed, slowly and uniformly inject equal volumes of high-concentration PEDV IgY purified antibody and zein core solution into container through dual-channel injection pump, mix thoroughly, and prepare translucent, slightly opalescent PEDV IgY colloidal dispersion; after centrifuging the nanoparticle suspension, collect the precipitate, resuspend in PBS (pH 7.0) and then sonicate to obtain PEDV IgY Zein / CMCS core-shell nanoparticle suspension.

[0019] Preferably, the PEDV virus solution is inactivated at 4°C for 12 h with a final concentration of 0.05% β-propiolactone, and then the β-propiolactone is hydrolyzed in a 37°C water bath for 2 h. This β-propiolactone is used as the antigen phase and emulsified with white oil adjuvant at a ratio of 1:2 to 3 to prepare a water-in-oil PEDV inactivated vaccine.

[0020] Preferably, healthy laying hens aged 30-35 weeks with an egg production rate of not less than 90% are selected, and the vaccine is administered intramuscularly at a dose of 1.0 mL / bird. After the first immunization, three booster immunizations are administered at 21-day intervals via the same dose to the wing root and leg muscles. High-immunity eggs are collected 14-35 days after the third immunization.

[0021] Preferably, the pH of the egg yolk aqueous extract is slowly and precisely adjusted to 4.8 using dilute hydrochloric acid for acidification. After acidification, octanoic acid with a final concentration of 0.2% is added while stirring to purify IgY.

[0022] Preferably, the prepared zein core solution is a 1% (w / v) zein solution and the carboxymethyl chitosan (CMCS) shell solution is a 0.2% (w / v) CMCS solution.

[0023] Preferably, while the CMCS shell solution is stirred at 1,200 rpm at 2–8°C, equal volumes of high-concentration PEDV IgY purified antibody and Zein core solution are slowly and uniformly injected into the container using a dual-channel syringe pump to thoroughly mix with the CMCS shell solution. The total volume of the Zein core solution and PEDV IgY purified antibody is 1 / 10 of the CMCS shell solution.

[0024] Preferably, the PEDV neutralizing antibody titer in the prepared PEDV IgY Zein / CMCS core-shell nanoparticle suspension is not less than 1:128.

[0025] This invention also provides an in vitro evaluation method for the tolerance and release of the above-mentioned PEDV IgY Zein / CMCS core-shell nanoparticle suspension in simulated gastric acid and intestinal fluid.

[0026] In another aspect of the invention, the application of the above-mentioned PEDV IgY Zein / CMCS core-shell nanoparticle suspension in the prevention or treatment of porcine epidemic diarrhea is also provided.

[0027] Preferably, the PEDV IgY Zein / CMCS core-shell nanoparticle suspension is administered orally at a dose of 2 mL per head, once a day, for at least 3 consecutive days.

[0028] Preferably, the PEDV IgY Zein / CMCS core-shell nanoparticle suspension can be fed directly or mixed with feed to sows that are about to give birth or are lactating, thereby increasing the PEDV IgY content in their milk and providing piglets with high titers of maternal antibodies.

[0029] Preferably, the PEDV hyperimmune protein can be fed directly or mixed with feed to sows that are about to give birth or are lactating, thereby increasing the PEDV IgY content in milk and providing piglets with high titers of maternal antibodies.

[0030] Beneficial effects: This invention isolated a GⅡc type PEDV HLJ04 strain with strong clinical pathogenicity in piglets. Through multiple rounds of double-layer plaque purification and domestication, the HLJ04 P10 / PFU cell-adapted strain was obtained, with significantly improved cell adaptability and a virus titer reduced from 10 before purification. 5.5 TCID 50 / mL increased to 10 8.0 TCID 50 / mL. Based on this strain, a PEDV inactivated vaccine with high antigen content was prepared. Immunization of laying hens yielded PEDV hyperimmune eggs with high and medium neutralizing antibody titers. High-purity PEDV IgY antibodies were obtained by purification using the octanoic acid-ammonium sulfate method. Using zein / carboxymethyl chitosan (Zein / CMCS) core-shell nanoparticle coating technology, a gastric acid-resistant PEDV IgY specific egg yolk antibody was developed. This antibody can resist the strong acid environment of the pig stomach and the destruction by pepsin. After being fully released in the intestine, it has good preventive and therapeutic effects against PEDV infection. Biological Preservation

[0031] Classification and nomenclature: The GⅡc type PEDV HLJ04 P10 / PFU cell-adapted strain of Porcineepidemic diarrhea virus purified by plaque was deposited at the China Center for Type Culture Collection on October 29, 2025, with accession number CTCC NO:V202576. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] Figure 1 This is a cytopathic image of PEDV HLJ04 strain in Example 1 of the present invention; Figure 2 This is an IFA identification image of PEDV HLJ04 strain from Example 1 of this invention; Figure 3 This is a transmission electron microscope image of PEDV HLJ04 strain from Example 1 of the present invention; Figure 4 This is the phylogenetic tree of the S gene of PEDV HLJ04 strain in Example 2 of the present invention; Figure 5 This is a comparison of phagocytic plaques of PEDV HLJ04 (left) and HLJ04 P10 / PFU (right) strains in Example 3 of this invention; Figure 6 This is a virus proliferation curve of PEDV HLJ04 P10 / PFU strain in Example 3 of the present invention; Figure 7 This is a graph showing the weight change of piglets in different experimental groups in Example 7 of the present invention. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0035] All chemical reagents used in the embodiments of this invention are of analytical grade.

[0036] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. Unless otherwise specified, the experimental methods described in this invention are conventional methods; and unless otherwise specified, the biological materials described are commercially available.

[0037] Example 1: Isolation and Identification of PEDV HLJ04 strain 1.1 Experimental Materials and Main Reagents 1.1.1 Cells and Samples Vero E6 cells were purchased from ATCC and preserved by the Henan Provincial Animal Disease and Public Health Engineering Research Center. Small intestinal tissue and contents of PEDV-infected piglets were collected from a pig farm in Qiqihar City, Heilongjiang Province. The affected piglets mainly exhibited typical symptoms such as severe diarrhea, dehydration, and vomiting. The tissue was tested by Luoyang Zhongke Gene Testing and Diagnostic Center Co., Ltd. and stored at -80℃.

[0038] 1.1.2 Main Reagents EasyPure ® Viral DNA / RNA Kit nucleic acid extraction kit was purchased from Beijing TransGen Biotech Co., Ltd.; MightyScript Plus first-strand cDNA synthesis Master Mix (genomic DNA removed) reverse transcription kit, 2×FidCycle Fast high-fidelity PCR Mix (containing blue dye), and low-melting-point agarose were all purchased from Sangon Biotech (Shanghai) Co., Ltd.; DMEM high-glucose medium, FBS fetal bovine serum, and 0.25% trypsin cell digestion solution were all purchased from Gibco; FITC-labeled goat anti-mouse IgG (H+L) secondary antibody was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; trypsin (derived from porcine pancreas) and β-propiolactone inactivator were purchased from Sigma-Aldrich; PEDV S protein mouse monoclonal antibody was prepared, identified, and preserved by the Henan Provincial Animal Disease and Public Health Engineering Research Center; poultry oil-in-water compound adjuvant was provided by Luoyang Saiwei Biotechnology Co., Ltd.; other chemical reagents were all of analytical grade.

[0039] 1.2 Isolation and Identification of Viruses 1.2.1 Virus isolation After thawing the pathogenic material at room temperature, it was weighed and added to serum-free DMEM pre-cooled at 2-8°C with 1000 IU / mL of penicillin and streptomycin at a weight (g) to volume (mL) ratio of 1:10. The mixture was then ground and repeatedly frozen and thawed three times. After centrifugation at 3000 r / min and 4°C for 30 min, the supernatant was filtered through a 0.22 μm filter for sterilization. Trypsin was added to the supernatant to a final concentration of 0.3 μg / mL and incubated at room temperature for 10 min to obtain the inoculation sample.

[0040] Vero E6 cells were revived and cultured in 10% FBS DMEM complete medium, and then treated with 0.25% trypsin digestion solution before cell counting at a density of 3.0 × 10⁻⁶ cells / cells. 5Cells were seeded into 6-well plates. On the second day, when the cell density reached 80%–90%, the culture medium was discarded, and the cells were washed three times with PBS. Samples were then seeded at 0.5 mL / well and incubated at 37°C with 5% CO2 for 1 h, shaking the plate every 15 min. After incubation, the seeding medium was discarded, and the medium was replaced with PEDV DMEM cell maintenance medium (0.3 μg / mL trypsin + 0.3% trypsin-phosphate broth + 0.02% yeast extract + DMEM) for continued culture. Cytopathic effects were observed daily. 96 h after seeding, the 6-well plates were freeze-thawed at -80°C, centrifuged to remove cell debris, and 1 mL / well of the supernatant was seeded into Vero E6 cells for blind passage. After blind passage to passage 5, Vero E6 cells exhibited typical pathological changes such as rounding, aggregation, and syncytia (e.g., ...). Figure 1 PEDV virus was successfully isolated and named PEDV HLJ04 strain, considered as the P0 generation virus. The P0 generation virus was inoculated into cells in T75 flasks for amplification culture to establish a seed batch (P1 generation). After aliquoting, the virus was stored at -80℃, and the virus titer was determined to be 10. 5.5 TCID 50 / mL.

[0041] 1.2.2 Identification of the virus Cell slides were prepared by inoculating Vero E6 cells with PEDV HLJ04 strain. Cells were fixed in acetone-methanol fixative (acetone:methanol = 1:1) pre-chilled at -20°C. The isolated virus was identified by indirect immunofluorescence (IFA) using a mouse monoclonal antibody against the PEDV S protein as the primary antibody and FITC-labeled goat anti-mouse IgG (H+L) as the secondary antibody. IFA results showed that specific green fluorescent positive cells appeared in PEDV-infected Vero E6 cells, while no green fluorescent positive cells were observed in Vero E6 cells uninfected with clinical PEDV isolates. (See attached table). Figure 2 Simultaneously, PEDV virus droplets, after centrifugation to remove cell debris, were placed on a copper grid with a supporting membrane and stained with phosphotungstic acid for negative staining. Transmission electron microscopy revealed the typical appearance of coronavirus particles; the virus particles varied in size, ranging from 80 nm to 120 nm in diameter, with clearly visible spikes on the outermost layer. Figure 3 .

[0042] Example 2: Genetic evolution analysis of PEDV HLJ04 strain Nucleic acid was extracted from PEDV strain HLJ04 according to the instructions of the nucleic acid extraction kit, and cDNA was obtained by reverse transcription. Primers for S gene PCR amplification were synthesized based on the conserved region sequences of PEDV NSP16, S, and ORF3 genes (Table 1). The reaction system consisted of: 25 μL of 2×FidCycle Fast high-fidelity PCR Mix (containing blue dye), 2 μL each of PEDV-NSP16-F1 / PEDV S-F2 (10 μM) and PEDVS-R1 / PEDV ORF3-R2 (10 μM), 5 μL of cDNA, and 16 μL of ddH2O, for a total of 50 μL. The reaction conditions were: pre-denaturation at 98℃ for 30 sec, denaturation at 98℃ for 10 sec, annealing at 60℃ for 10 sec, extension at 72℃ for 3 min (35 cycles of denaturation, annealing, and extension), final extension at 72℃ for 10 min, and ending the reaction at 25℃ for 5 min. After PCR products were subjected to 1% agarose gel electrophoresis, the target gene fragment was excised. The gel return products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were processed and edited using DNASTAR Lasergene software. Referring to a representative PEDV strain, the S gene was subjected to genetic evolution analysis and nucleotide homology alignment using Mega 7.0 software.

[0043] Table 1 Primers for PEDV S whole-genome amplification

[0044] Genetic evolutionary analysis results indicate that the PEDV HLJ04 strain in this invention belongs to the GIIc clade within the GII genotype, such as... Figure 4 PEDV has become the dominant circulating branch in my country over the past two years, with high morbidity and mortality rates in piglets. Nucleotide homology comparison results showed that the S gene nucleotide homology of PEDV strain HLJ04 with 29 selected representative PEDV strains from both domestic and international sources was 93.1%–99.0%. The S gene nucleotide homology with currently used clinical PEDV vaccine strains—GⅠb branch CV777, GⅡb branch AJ1102-R and LW / L, and GⅡa branch XJ-DB2—was 93.3%, 96.9%, 96.6%, and 97.7%, respectively, with amino acid homology of 92.0%, 97.3%, 96.0%, and 97.2%, respectively. The homology with existing vaccine strains was relatively low.

[0045] Example 3: Cell adaptation culture and plaque purification of PEDV HLJ04 strain To improve the cell adaptability and viral titer of PEDV HLJ04 strain, Vero E6 cells were purified and passaged using a double plaque method after infection with P1 generation virus. The specific method is as follows: (1) Vero E6 cells were seeded into six-well plates and cultured overnight at 37°C with 5% CO2. On the second day, when the cells reached approximately 90% confluence, the culture medium in the six-well plates was discarded, and different dilutions of HLJ04 virus solution were inoculated at 500 μL / well. The plates were incubated at 37°C for 1 h, with gentle shaking every 15 min. After incubation, the inoculum was discarded, and 3 mL / well was added as the first layer of covering material (PEDV cell maintenance medium containing 0.6% low-melting-point agarose). After the covering material solidified, the plates were cultured further.

[0046] (2) After the first layer of covering material is added, add the second layer of covering material at 3 mL / well for 48–72 h and continue culturing. The formula for the second layer of covering material is: 7.5% NaHCO3 (340 μL) + 0.33% Neutral Red (240 μL) + 1.2% Low Melting Point Agarose (10 mL) + 2×PEDV Cell Maintenance Medium (10 mL).

[0047] (3) 12 to 24 h after the second layer of covering material is added, observe the morphology of viral plaques, select relatively large viral plaques from the high dilution inoculation wells, pick them up with the pipette tip and inoculate them into 12-well plates with 80% to 90% confluence of Vero E6 cells for culture.

[0048] (4) When cytopathic effects appear 48–96 h after inoculation, the cell culture supernatant is collected after one freeze-thaw cycle, thus completing the first generation of plaque purification. Steps (1) to (5) are repeated to complete the 10th generation of plaque purification, which is named PEDV HLJ04 P10 / PFU strain. After purification, the virus is inoculated into Vero E6 cells for propagation, dispensed into 0.5 mL / EP tubes, and stored at -80°C. Through monolayer plaques, it can be observed that the viral plaques are uneven in size and small before purification, while the plaques are larger and more uniform in size after purification. The results are shown in […]. Figure 5 .

[0049] Serial dilutions of PEDV HLJ04 P10 / PFU strain were performed 10-fold, and Vero E6 cells were inoculated into 96-well plates to determine viral titers. Each dilution was performed in 8 replicates. The viral titer was determined by 10-10 7.5 TCID 50 / mL, significantly higher than 10 before purification. 5.5 TCID 50 / mL, indicating that plaque purification and continuous passage in cells improved the cell adaptability of the virus. The purified virus was seeded into Vero E6 cells in 6-well plates at MOIs of 0.001 and 0.01, respectively. Cytopathic effects were observed daily after seeding. Virus was harvested at 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h post-seeding. After one freeze-thaw cycle, cell debris was removed by centrifugation, and the viral titer at different time points was measured. Growth curves of the PEDV HLJ04 P10 / PFU strain were plotted. The results showed that the viral titer peaked at 48 h after seeding with an MOI of 0.01 and at 72 h after seeding with an MOI of 0.001, followed by a slow decrease in viral titer. Both seeding doses achieved viral titers of 10. 8.0 TCID 50 / mL, results are shown in Figure 6 .

[0050] Example 4: Preparation and purification of PEDV egg yolk antibody 4.1 Vaccine preparation and testing results Following the inoculation dosage and virus harvesting time in Example 3, PEDV HLJ04 P10 / PFU strain was used to infect cells, and the virus fluid was harvested. β-propiolactone was added to a final concentration of 0.05% for inactivation at 4°C for 12 h, followed by hydrolysis of β-propiolactone in a 37°C water bath for 2 h. No cytopathic effects were observed in three blind passages of the inactivated virus fluid in Vero E6 cells, indicating that the inactivation test was successful. Using the inactivated virus as the antigen aqueous phase and a white oil adjuvant as the oil phase, a water-in-oil PEDV inactivated vaccine was prepared by emulsification at a 1:2 ratio of aqueous phase to oil phase. After passing the dosage form, stability, and sterility tests according to the Chinese Veterinary Pharmacopoeia (2020 edition), the prepared inactivated vaccine was used to immunize laying hens.

[0051] 4.2 Immunization of laying hens and purification of egg yolk antibodies Healthy Hy-Line Brown hens aged 30-35 weeks with no history of disease and an egg production rate of not less than 90% were selected. The vaccine was administered via intramuscular injection at multiple sites in the breast at a dose of 1.0 mL / bird. Three booster immunizations were administered 21 days after the initial immunization, via intramuscular injection at the wing root and leg muscles. Hyperimmune eggs collected 14-35 days after the last immunization were used to purify PEDV yolk antibodies using the caprylic acid-ammonium sulfate method, as follows: (1) Preparation of egg yolk aqueous extract High-immunity eggs with no visible contamination were disinfected by soaking in a 0.1% benzalkonium chloride solution at 42°C for 15 minutes. The eggshells were broken to remove the egg white and chalaza, and the yolks were collected. The yolks were washed three times with sterile ultrapure water to thoroughly remove any residual egg white from the surface. Seven times the volume of the yolks were added to pre-cooled sterile deionized water, stirred thoroughly, and centrifuged at 8000 r / min for 30 minutes at 4°C. The supernatant was used as the yolk extract.

[0052] (2) Acidification of aqueous extract and extraction with octanoic acid Under continuous stirring at 2–8°C, the pH of the egg yolk aqueous extract was slowly and precisely adjusted to 4.8 with 1M hydrochloric acid to acidify the extract. While continuing stirring, a 10% caprylic acid solution was added dropwise to the acidified extract to a final concentration of 0.2%. After adding caprylic acid, stirring was continued for 30 min. The extract was then centrifuged at 8000 r / min for 30 min at 4°C. The supernatant containing purified IgY was collected, and the pH of the extract was immediately adjusted back to 7.0 with 1M sodium hydroxide solution.

[0053] (3) Concentration and dialysis Slowly add saturated ammonium sulfate solution to a final concentration of 45% to the purified IgY supernatant, let stand at room temperature for 3 h, centrifuge at 8000 r / min for 30 min at 4℃, discard the supernatant, and dissolve the precipitate with an equal volume of sterile PBS to the egg yolk extract. Place the extracted IgY solution into a dialysis bag, put it in a beaker containing PBS, and dialyze overnight at 2–8℃ on a magnetic stirrer to remove salts and small molecule impurities.

[0054] (4) Ultrafiltration concentration The dialyzed IgY solution was concentrated by ultrafiltration using a membrane with a molecular weight cutoff of 30 KD to 1 / 8 of its volume, yielding a PEDV IgY antibody solution, which was stored at 2–8°C.

[0055] 4.3 Determination of PEDV neutralizing antibody titer (1) Plating and virus dilution: Well-grown Vero E6 cells were digested with 0.25% trypsin and counted, then plated at 2×10⁻⁶ cells per cell line. 4 Cells were added to 96-well cell culture plates at a density of 100 μL per well and incubated at 37°C in a 5% CO2 incubator. The HLJ04 strain was diluted to a working concentration of 200 TCID50 with PEDV maintenance medium. 50 / 0.1mL.

[0056] (2) Virus neutralization: PEDV IgY antibody solution and unpurified egg yolk extract were serially diluted 2 times to 1:4096 using PEDV maintenance solution. Each diluted antibody solution was mixed with an equal volume of virus solution at working concentration and placed at 37°C for 1 hour to neutralize, shaking 2 to 3 times during the process.

[0057] (3) Culture and observation: The neutralized samples were inoculated into 96-well cell culture plates that had grown into a well-formed monolayer. Eight wells were added for each dilution, with 100 μL added to each well. Virus control wells and normal cell control wells were also included. The 96-well plates were cultured and observed daily. The results were determined 96 hours after sample inoculation.

[0058] (4) Result determination: There should be no cytopathic effect in the normal cell control wells, and all virus control wells should show cytopathic effect; otherwise, the result is invalid. Observe and count the cytopathic effect in the inoculation wells of different sample dilutions, and calculate the neutralizing antibody titer according to the Reed-Muench method.

[0059] The neutralizing antibody titer of PEDV in the egg yolk extract before purification was determined to be 1:1024 using the above method, and the neutralizing antibody titer of the purified PEDVgY antibody solution was 1:512. Both the samples before and after purification contained high titers of PEDV neutralizing antibodies.

[0060] Example 5: Acid-resistant coating of PEDV egg yolk antibody PEDV egg yolk antibody (IgY) has been proven to have immunoprophylactic effects against PEDV, alleviating clinical symptoms in sick piglets and reducing mortality in challenged piglets. However, egg yolk antibody (IgY) is essentially a protein, which is easily degraded and destroyed in the highly acidic and proteolytic environment of the piglet's stomach. Oral delivery requires coating. Various coating technologies can achieve gastric protection while maintaining a liquid state. This invention utilizes zein / carboxymethyl chitosan (Zein / CMCS) core-shell nanoparticle technology to coat PEDV IgY antibody within nanoparticles, preparing a PEDV IgY nanoemulsion oral solution that is resistant to piglet gastric juice, fully released in the small intestine, and has good colloidal stability. This solution is used for the prevention and treatment of PEDV in piglets via gavage.

[0061] 5.1 Solution Preparation (1) Zein core solution Weigh a certain amount of zein powder and dissolve it in a 70% (v / v) aqueous ethanol solution. Stir magnetically at room temperature for at least 2 hours until the solution becomes clear and transparent. Prepare a 1% (w / v) Zein core solution as the organic phase.

[0062] (2) Carboxymethyl chitosan (CMCS) shell solution A certain amount of carboxymethyl chitosan powder was weighed and dissolved in ultrapure water. The solution was magnetically stirred at room temperature for at least 1 hour until completely dissolved to prepare a 0.2% (w / v) CMCS solution. The pH of the CMCS solution was adjusted to 5.0–5.5 with dilute acetic acid or NaOH solution and used as the aqueous phase. This pH environment is most favorable for electrostatic recombination between positively charged Zein and negatively charged CMCS.

[0063] 5.2 Preparation of Zein core nanoparticles A coated PEDV IgY colloidal solution was prepared using an antisolvent method. A certain volume of CMCS shell solution was placed in a sterile container. While stirring at 1200 rpm at 2–8°C, equal volumes of high-concentration PEDV IgY purified antibody and Zein core solution were slowly and uniformly injected into the container using a dual-channel syringe pump, ensuring thorough mixing with the CMCS shell solution. The total volume of the Zein core solution and PEDV IgY purified antibody was 1 / 10 of the CMCS shell solution. After injection, high-speed stirring continued for 30–60 min to further stabilize the nanoparticle structure and ensure sufficient diffusion and evaporation of ethanol. A translucent, slightly opalescent PEDV IgY colloidal dispersion was successfully prepared.

[0064] 5.3 Post-processing and purification To obtain pure nanoparticles and improve product quality, ethanol, a small amount of free unencapsulated antibody, and excess CMCS must be removed from the system. The above nanoparticle suspension was centrifuged at 15,000 rpm for 30 min at 4°C. The supernatant was discarded, and the nanoparticle precipitate was resuspended in PBS (pH 7.0) of equal volume to the original PEDV IgY. The nanoparticles were then redispersed by short-term low-frequency sonication, resulting in the EDV IgY Zein / CMCS core-shell nanoparticle suspension.

[0065] 5.4 Dilution and Storage The PEDV neutralizing antibody titer of the EDV IgY Zein / CMCS core-shell nanoparticle suspension was determined according to the method in Example 4.3. The suspension was diluted with sterile PBS (pH 7.0) to a neutralizing antibody titer of at least 1:128 to obtain the PEDV IgY nanoemulsion oral solution, which was stored at 2–8°C for 3–4 weeks. For long-term storage, the nanoparticle precipitate was resuspended in an appropriate volume of PBS (pH 7.0–7.4), a lyophilization protectant (such as 5% trehalose or mannitol) was added, and the mixture was pre-frozen and then freeze-dried to obtain a nanoparticle powder that is easy to store and transport. When using, the powder was resuspended in sterile PBS (pH 7.0–7.4) to a neutralizing antibody titer of at least 1:128.

[0066] Example 6: In vitro evaluation of gastric acid resistance and intestinal release simulation To evaluate the ability of the nanoemulsion oral solution prepared in this invention to tolerate piglet gastric juice, maintain the neutralizing activity of PEDV IgY, and be fully released in the small intestine, in vitro experiments were conducted to evaluate its resistance to gastric acid and its intestinal release effect.

[0067] 6.1 Preparation of Artificial Gastric and Small Intestinal Fluids for Piglets (1) Preparation of artificial gastric juice for piglets Dissolve 0.2 g NaCl in 800 mL of sterile ultrapure water, add 2.0 mol / L dilute hydrochloric acid to adjust the pH to 2.5, then gently stir and slowly add 3.2 g pepsin until fully dissolved, and finally bring the volume to 1000 mL with sterile ultrapure water. Prepare and use immediately.

[0068] (2) Preparation of artificial small intestine fluid for piglets Dissolve 6.8 g KH2PO4 in 800 mL of sterile ultrapure water, adjust the pH to 6.8 by adding 1.0 mol / L NaOH dropwise, add 5.0 g of porcine bile salts, and then slowly add 10.0 g of porcine pancreas extract (mainly a mixture of trypsin, pancreatic amylase and pancreatic lipase) while gently stirring. Dissolve thoroughly, and finally bring the volume to 1000 mL with sterile ultrapure water. Prepare and use immediately.

[0069] 6.2 In vitro evaluation of resistance to gastric juice and intestinal release (1) Stomach simulation stage: The same volume of the PEDV IgY antibody nanoparticle suspension prepared in Example 5 was added to the artificial gastric fluid of piglets that was oscillating at 37°C, and the gastric emptying time was simulated and incubated for 2 hours.

[0070] (2) Intestinal simulation stage: Take out the sample after gastric juice treatment, centrifuge at high speed and collect the precipitate, resuspend it to the original volume using sterile PBS (pH 7.0-7.4), and add an equal volume to the artificial small intestine of piglets at 37℃ constant temperature shaking for 4-6 hours.

[0071] (3) Determination of the retention rate of neutralizing activity of PEDV IgY antibody The neutralizing antibody titer of the above mixture was determined according to the method in Example 4. A control was set up, using PEDV IgY antibody extract untreated with Zein / CMCS core-shell nanoparticle technology, to compare the retention rate of neutralizing activity. Before treatment with piglet artificial gastric and small intestinal fluids, the PEDV neutralizing antibody ratio of the PEDV IgY antibody nanoparticle suspension was 1:256, and after treatment it was 1:218, with a neutralizing activity retention rate of 85.16%, significantly higher than that of the untreated PEDV IgY antibody extract. The latter, after treatment with piglet artificial gastric and small intestinal fluids, showed a decrease in neutralizing antibody ratio from 1:256 to 1:20.1, with a neutralizing activity of only 7.85% (see Table 2). The results indicate that the highly acidic environment in the piglet stomach denatures the IgY antibody protein, causing irreversible changes in its three-dimensional structure and resulting in the loss of its ability to recognize and bind to the virus. Simultaneously, the pepsin-rich gastric fluid can enzymatically degrade the antibody into small peptide fragments, thereby completely destroying its function. PEDV IgY antibodies processed with Zein / CMCS core-shell nanoparticle technology can safely pass through the stomach and be effectively released in the small intestine where PEDV adheres and proliferates, thus exerting an effective preventive and therapeutic effect on PEDV infection.

[0072] Table 2. Retention rate of neutralizing activity of PEDV IgY antibody

[0073] Example 7 Evaluation of the preventive and therapeutic effects of gastric acid-resistant PEDV egg yolk antibodies on piglets Twenty-five healthy newborn piglets were born to sows that tested negative for PEDV, PoRV, TGEV, PDCOV, ASFV, PRRSV, PRV antigen, and PEDV antibody. They were fed with piglet-specific milk powder and randomly divided into five groups of five piglets each. Group 1 was the prevention group; piglets aged 1-3 days were administered 2 mL / head of acid-resistant PEDV egg yolk antibody daily via gavage, and infected with PEDV HLJ04 strain at 4 days of age. Group 2 was the treatment group; piglets were infected with PEDV virus at 4 days of age, and administered 2 mL / head of acid-resistant PEDV egg yolk antibody daily via gavage from 5-7 days of age. Group 3 was the prevention and treatment group; piglets aged 1-3 days were administered 2 mL / head of acid-resistant PEDV egg yolk antibody daily via gavage for prevention, infected with PEDV virus at 4 days of age, and treated with 2 mL / head of acid-resistant PEDV egg yolk antibody daily via gavage from 5-7 days of age. Group 4 was the virus infection control group; piglets were infected with PEDV virus at 4 days of age. Group 5 was the blank control group; piglets at 4 days of age were orally administered the same volume of DMEM culture medium. The infection dose of PEDV HLJ04 strain for piglets in groups 1-4 was 10... 5.0 TCID 50 The volume was 2 mL per piglet. During the experiment, piglets in each group were weighed every other day. After the challenge, the incidence and mortality of piglets in each group were recorded until the piglets reached 14 days of age.

[0074] During the experiment, piglets in the virus-infected control group began to show diarrhea as early as 12 hours after infection, which became watery diarrhea at 24 hours, accompanied by vomiting. Death began at 36 hours, with a survival rate of 40% on day 4 post-infection and all piglets dying on day 6. Piglets in the prevention group, treatment group, preventive-treatment group, and blank control group showed no clinical symptoms of diarrhea or vomiting throughout the experiment, with a survival rate of 100%. This indicates that the acid-resistant PEDV egg yolk antibody provided effective anti-infection protection for piglets. The results are shown in Table 3. Piglet weight changes showed that the prevention group, treatment group, and preventive-treatment group consistently increased in weight throughout the experimental period, with no difference from the blank control group; while the virus-infected control group experienced a continuous decrease in weight after infection until death. (See Table 3 for details.) Figure 7 Experimental results show that acid-resistant PEDV egg yolk antibodies can provide 100% protection against PEDV infection in piglets.

[0075] Table 3 Survival rates of piglets in different experimental groups

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the technical solution of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for detecting PEDV strain GIIc, characterized in that, The strain in question is PEDV HLJ04 P10 / PFU, with accession number CTCC NO:V202576.

2. The method for preparing the GIIc type PEDV virus strain as described in claim 1, comprising the following steps: (1) GⅡc type PEDV positive tissue samples were ground, frozen and thawed, and the supernatant was filtered and sterilized before being inoculated into cells for virus isolation. After inoculation, the samples were replaced with PEDV DMEM cell maintenance medium and continuously blindly passaged until Vero E6 cells showed typical lesions such as rounding, aggregation, and syncytia. The S gene of the isolates was sequenced and genetic evolution was analyzed. (2) The virus plaques were continuously purified and passaged using double plaques. After different dilutions of HLJ04 virus solution were inoculated into Vero E6 cells, the first layer of covering material was added. After further culture, the second layer of covering material was added. The morphology of the viral plaques was observed, and the viral plaques were selected for inoculation into Vero E6 cells for cell passage.

3. The preparation method according to claim 2, characterized in that, The isolates were identified by indirect immunofluorescence and phosphotungstic acid negative staining followed by transmission electron microscopy. PEDV was maintained in DMEM cell maintenance medium containing 0.3 μg / mL trypsin, 0.3% trypsin-phosphate broth, and 0.02% yeast extract.

4. The preparation method according to claim 2, characterized in that, The first layer of plaque purification was PEDV DMEM cell maintenance medium containing 0.6% low-melting-point agarose. The second layer of purification medium was prepared by mixing 340 μL of 7.5% NaHCO3 solution, 240 μL of 0.33% neutral red solution, 10 mL of 1.2% low-melting-point agarose, and 10 mL of 2-fold PEDV DMEM cell maintenance medium. The second layer of purification medium was added 48–72 h after the first layer of purification medium. After the second layer of purification medium was added, the cells were incubated for another 12–24 h for observation. Large plaques were selected and seeded into Vero E6 cells for acclimatization and passage. Plaque purification was carried out for at least 10 passages.

5. The application of the preparation method for the GIIc type PEDV virus strain as described in claim 1 or any one of claims 2-5 in the development of inactivated PEDV vaccines, live attenuated vaccines, and PED-related diagnostic reagents for pigs.

6. A method for preparing a gastric acid-resistant specific egg yolk antibody against GIIc type PEDV virus strain, comprising the following steps: (1) Vaccine preparation and immunization of laying hens: The PEDV HLJ04 P10 / PFU cell-adapted strain purified by plaque, with the preservation number CTCC NO:V202576, was inoculated into Vero E6 cells to prepare virus solution. After inactivation, water-in-oil type PEDV inactivated vaccine was prepared. Healthy laying hens during the laying period were immunized with the inactivated vaccine. After the first immunization, three booster immunizations were carried out every 21 days. High-immunity eggs were collected 14 to 35 days after the last immunization. (2) Purification of egg yolk antibodies: After disinfection by soaking in 0.1% benzalkonium chloride, the eggs were broken and the yolks were collected. Sterile deionized water was added at 7 times the volume of the yolks and the mixture was stirred and centrifuged to obtain the supernatant as the egg yolk aqueous extract. The egg yolk aqueous extract was acidified with dilute hydrochloric acid, and then caprylic acid was added while stirring to purify IgY. The pH of the purified IgY supernatant was adjusted back to 7.0, and ammonium sulfate solution was added to precipitate the IgY antibody. The IgY antibody precipitate was dissolved in sterile PBS, placed in a dialysis bag and dialyzed overnight to remove salt and small molecule impurities. The dialyzed IgY solution was concentrated by ultrafiltration using a membrane with a molecular weight cutoff of 30 KD to obtain the PEDV IgY antibody solution. (3) Acid-resistant coating of antibody: Prepare zein organic phase as core solution, prepare carboxymethyl chitosan aqueous phase, and adjust pH to 5.0-5.5 shell solution; while stirring CMCS shell solution at high speed, slowly and uniformly inject equal volumes of high-concentration PEDV IgY purified antibody and Zein core solution into container through dual-channel injection pump, mix thoroughly, and prepare translucent, slightly opalescent PEDV IgY colloidal dispersion; after centrifuging the nanoparticle suspension, collect the precipitate, resuspend in PBS at pH 7.0 and then sonicate to obtain PEDV IgY Zein / CMCS core-shell nanoparticle suspension.

7. The preparation method according to claim 6, characterized in that, PEDV virus solution was inactivated at 4°C for 12 h with a final concentration of 0.05% β-propiolactone, followed by hydrolysis of β-propiolactone in a 37°C water bath for 2 h. This was used as the antigen phase and emulsified with white oil adjuvant at a ratio of 1:2 to 3 to prepare a water-in-oil PEDV inactivated vaccine. Healthy laying hens aged 30 to 35 weeks with an egg production rate of not less than 90% were selected and injected intramuscularly at a dose of 1.0 mL / bird. After the first immunization, three booster immunizations were administered at 21-day intervals via the same dose to the wing root and leg muscles. High-immunity eggs were collected 14 to 35 days after the third immunization.

8. The preparation method according to claim 6, characterized in that, The pH of the egg yolk aqueous extract was slowly and precisely adjusted to 4.8 using dilute hydrochloric acid for acidification. After acidification, octanoic acid with a final concentration of 0.2% was added while stirring to purify IgY. The prepared zein core solution was a 1% (w / v) zein solution, and the carboxymethyl chitosan shell solution was a 0.2% (w / v) CMCS solution. While the carboxymethyl chitosan shell solution was stirred at 1,200 rpm at 2–8°C, equal volumes of high-concentration PEDV IgY purified antibody and zein core solution were slowly and uniformly injected into the container using a dual-channel syringe pump and thoroughly mixed with the carboxymethyl chitosan shell solution. The total volume of the zein core solution and PEDV IgY purified antibody was 1 / 10 of that of the carboxymethyl chitosan shell solution. In the prepared PEDV IgY Zein / CMCS core-shell nanoparticle suspension, the PEDV neutralizing antibody titer was not less than 1:

128.

9. The application of the preparation method as described in claim 6 in the prevention or treatment of porcine epidemic diarrhea.

10. The application as described in claim 9, characterized in that, PEDV IgY Zein / CMCS core-shell nanoparticle suspension can be fed directly or mixed with feed to sows nearing farrowing or lactating, increasing the PEDV IgY content in milk and providing piglets with high titers of maternal antibodies.

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