Recombinant porcine coronavirus

Abstract

The invention relates to the field of animal health. Specifically, the invention relates to a recombinant porcine coronavirus, in particular to a recombinant porcine epidemic diarrhea virus (PEDV), and the genome of the recombinant porcine coronavirus comprises deletion of a complete non-structural protein 2 (NSP2) coding sequence. In addition, the invention provides an immunogenic composition comprising the recombinant porcine coronavirus of the invention and uses thereof.

Description

Technical Field

[0001] The present invention relates to the field of animal health. Specifically, the present invention relates to a recombinant porcine coronavirus, in particular a recombinant porcine epidemic diarrhea virus (PEDV), whose genome includes a complete deletion of the non-structural protein 2 (NSP2) coding sequence. In addition, the present invention provides an immunogenic composition comprising the recombinant porcine coronavirus of the present invention and its use. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is an enveloped, positive-sense, single-stranded RNA virus that causes acute diarrhea, vomiting, and dehydration in pigs. In pigs three weeks of age and younger, clinical signs of PEDV infection (including acute watery, diarrhea, vomiting, and dehydration) can be seen as early as 24 hours after infection, resulting in up to 100% mortality. In addition, gross and histological changes in the intestine of animals infected with PEDV may result in gross pathological damage to the small intestine.

[0003] PEDV was first discovered in Europe, but is a growing problem in many Asian countries, including South Korea, China, Japan, the Philippines, and Thailand. PEDV has been present in the United States since 2013, and the economic impact of PEDV infection has been significant.

[0004] Therefore, there is an ongoing need to develop vaccines that can protect pigs from PEDV-associated diseases. Summary of the invention

[0005] In one aspect, the present invention provides a recombinant porcine coronavirus comprising a deletion of the complete nonstructural protein 2 (NSP2) coding sequence in its genome.

[0006] In one aspect, the present invention provides a recombinant porcine coronavirus, which is

[0007] i) the recombinant PEDV deposited with the CCTCC under accession number CCTCC V202263 on August 10, 2022, or any of its progeny or descendants; or

[0008] ii) a recombinant PEDV, whose genomic sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity with the recombinant PEDV deposited with the CCTCC under the accession number CCTCC V202263 on August 10, 2022.

[0009] In one aspect, the present invention provides an isolated nucleic acid encoding a recombinant porcine coronavirus of the present invention.

[0010] In one aspect, the invention provides a vector comprising a nucleic acid of the invention.

[0011] In one aspect, the present invention provides an immunogenic composition comprising the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention, or the vector of the present invention.

[0012] In one aspect, the present invention provides a use of the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention or the vector of the present invention in preparing an immunogenic composition, wherein the immunogenic composition is used for

[0013] i) Immunization of pigs;

[0014] ii) eliciting an immune response against porcine coronavirus in pigs;

[0015] iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or

[0016] iv) Reducing mortality caused by porcine coronavirus infection in pigs.

[0017] In one aspect, the present invention provides a use of the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention or the vector of the present invention in preparing an immunogenic composition, wherein the immunogenic composition is used for

[0018] i) reducing or preventing clinical symptoms or disease caused by PEDV infection in piglets that are to be suckled by sows to which the immunogenic composition has been administered; and / or

[0019] ii) reducing mortality caused by PEDV infection in piglets suckled by sows to which the immunogenic composition has been administered.

[0020] In one aspect, the present invention provides a method for

[0021] i) Immunization of pigs;

[0022] ii) eliciting an immune response against porcine coronavirus in pigs;

[0023] iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or

[0024] iv) reducing mortality caused by porcine coronavirus infection in pigs,

[0025] The method comprises administering the immunogenic composition of the invention to a pig.

[0026] In one aspect, the invention provides a method for i) reducing or preventing clinical symptoms or disease caused by PEDV infection in piglets, and / or ii) reducing mortality caused by PEDV infection in piglets, wherein the piglets are suckled by sows to which an immunogenic composition has been administered.

[0027] In one aspect, the present invention provides a method for attenuating porcine coronavirus, comprising deleting the entire nonstructural protein 2 (NSP2) coding sequence from its genome.

[0028] In one aspect, the present invention provides a method for producing a recombinant porcine coronavirus of the present invention, comprising

[0029] introducing the isolated nucleic acid of the invention or the vector of the invention into a host cell, and

[0030] Recovering the recombinant porcine coronavirus produced by the host cells.

[0031] In one aspect, the invention provides a method for distinguishing an animal infected with a porcine coronavirus from an animal vaccinated with an immunogenic composition of the invention, comprising:

[0032] a) obtaining a sample, and

[0033] b) Testing the sample in immunoassays and / or genomic profiling assays. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 .Strategies for rescuing the PEDV ZJU / G2 / 2013 strain with complete deletion of the NSP2 coding sequence.

[0035] Figure 2 .Methods for rescuing infectious viruses.

[0036] Figure 3 . The rescued rPEDV-delNsp2 virus was confirmed by indirect immunofluorescence (IFA) using PEDV-Nsp2 rabbit polyclonal antibody.

[0037] Figure 4 .RT-PCR identification of the rescued virus rPEDV-delNsp2.

[0038] Figure 5 .Plaque assay results of rPEDV-delNsp2.

[0039] Figure 6 .Growth curve of rPEDV-delNsp2 virus.

[0040] Figure 7 .24 hours after virus infection, the RNA synthesis amount of rPEDV-delNsp2 was significantly lower than that of the parental virus.

[0041] Figure 8 .24h after virus infection, the N protein expression of rPEDV-delNsp2 was significantly lower than that of the WT-PEDV-G2-ZJU infection group.

[0042] Fig. 9 .Morbidity and mortality following vaccination of piglets with various candidates.

[0043] Fig.10 .Clinical scores and survival rates in safety evaluation experiments.

[0044] Fig.11 .The virus shedding results were detected by qRT-PCR in the safety evaluation experiment.

[0045] Fig.12 .In the safety evaluation experiment, the PEDV antibody level in piglet serum at 14dpi was detected by CCIFA.

[0046] Fig.13 .In the safety evaluation experiment, PEDV antibodies in the serum of sows after exposure were detected by CCIFA.

[0047] Fig.14 .In the safety evaluation experiment, the PEDV antibody level in piglet serum was detected by ELISA.

[0048] Fig.15 . Construction and rescue of rSADS-CoV-Δnsp2. (A) Organization of the SADS-CoV “SeACoV-p10” genome (top). Details of the nsp2 gene before and after deletion are shown (bottom). (B) Detection of viral N protein in Vero cells infected with rSADS-CoV (as a control) or rSADS-CoV-Δnsp2 using IFA with anti-N polyclonal antibody 36 h after infection. Magnification = 200×. (C) Confirmation of nsp2 deletion in rSADS-CoV-Δnsp2. Sequencing results show fragments of the nsp2 deletion region from plasmids carrying infectious cDNA clones of rSADS-CoV-Δnsp2 (top) or RT-PCR amplification of the same region of viral RNA extracted from rSADS-CoV-Δnsp2-infected Vero cells (bottom). The corresponding amino acids are shown below. (D) Comparison of the growth kinetics of rSADS-CoV and rSADS-CoV-Δnsp2 in Vero cells. Cells were infected in triplicate with viruses at an MOI = 0.1. Cells were harvested at 2, 12, 24, 48 and 72 hpi and viral titers (TCID50 / ml) were determined in triplicate on Vero cells. DETAILED DESCRIPTION

[0049] Before describing various aspects of the present invention, it must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "protein" includes a plurality of proteins, reference to a "virus" refers to one or more viruses and equivalents thereof known to those skilled in the art, and so forth. The term "and / or" is intended to encompass any combination of items connected by the term, equivalent to listing all combinations individually. For example, "A, B and / or C" encompasses "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C". Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods, apparatus and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the viral strains, cell lines, vectors and methods reported in the publications, which may be used in conjunction with the present invention. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0050] The present invention is based on the surprising discovery that deletion of the NSP2 coding sequence of a porcine coronavirus such as PEDV results in attenuation of the virus and that the attenuated live virus may be suitable for providing effective protection against infection or challenge with a porcine coronavirus such as PEDV of pigs.

[0051] Therefore, in one aspect, the present invention provides a recombinant porcine coronavirus comprising a complete deletion of the non-structural protein 2 (NSP2) coding sequence in its genome.

[0052] The term "recombinant" refers to a porcine coronavirus that has been altered, rearranged or modified by genetic engineering. However, the term does not refer to changes in the polynucleotide or amino acid sequence caused by naturally occurring events such as spontaneous mutations.

[0053] In some embodiments, the deletion of the entire nonstructural protein 2 (NSP2) coding sequence is a complete deletion of the NSP2 gene.

[0054] In some embodiments, the virus is capable of infecting a host cell. The host cell may be a cell or cell line from a pig.

[0055] "Infection of host cells" as referred to herein specifically includes attachment of the virus to the host cell, entry of the virus into the cell, disassembly of the virion, replication and transcription of the viral genome, expression of viral proteins, and assembly and release of new infectious viral particles.

[0056] In some embodiments, the recombinant porcine coronavirus is attenuated. For example, the recombinant porcine coronavirus has lower virulence than the corresponding wild-type porcine coronavirus, which does not include a deletion of the entire nonstructural protein 2 (NSP2) coding sequence in its genome.

[0057] In some embodiments, the porcine coronavirus can be an alphacoronavirus, a betacoronavirus, or a deltacoronavirus.

[0058] Currently, six different porcine coronaviruses have been identified, among which porcine epidemic diarrhea virus (PEDV), porcine acute diarrhea syndrome-coronavirus (SADS-CoV), transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) belong to alpha coronaviruses; porcine hemagglutinating encephalomyelitis virus (PHEV) belongs to beta coronaviruses; and porcine delta coronavirus (PDCoV) belongs to delta coronaviruses.

[0059] In some embodiments, the porcine coronavirus can be selected from porcine epidemic diarrhea virus (PEDV), porcine acute diarrhea syndrome coronavirus (SADS-CoV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine delta coronavirus (PDCoV).

[0060] In some embodiments, the porcine coronavirus is PEDV. In some embodiments, the porcine coronavirus is genotype 2 PEDV.

[0061] It should be understood that the term "genotype" is equivalent to the term "genotype" frequently used in the literature in the context of PEDV. The terms "genotype 2a (G2a)" and "genotype 2b (G2b)" are well known to those skilled in the art. Through phylogenetic studies of the S gene, PEDV can be genetically divided into 2 groups: genotype 1 (G1; classical) and genotype 2 (G2; field epidemic or pandemic). Each genotype can be further divided into subgroups (1a and 1b; 2a and 2b). G2 includes global field isolates, which are further divided into 2a and 2b subgroups, responsible for previous local epidemic outbreaks in Asia and recent epidemic outbreaks in North America and Asia, respectively.

[0062] In some embodiments, the recombinant porcine coronavirus is derived from the PEDV-ZJU / G2 / 2013 strain.

[0063] The PEDV-ZJU / G2 / 2013 strain is a virulent strain isolated in Zhejiang Province, China in 2013. The genome sequence of the PEDV-ZJU / G2 / 2013 strain can be obtained from GenBank with accession number KU558701.1. The infectious clone of PEDV-ZJU / G2 / 2013 can be rescued according to the disclosure of Chinese patent application number: 202010162003.0 (published as CN111471709A).

[0064] In some embodiments, the porcine coronavirus is SADS-CoV.

[0065] In some embodiments, the recombinant porcine coronavirus is derived from the SADS-CoV-CH / GD-01 / 2017 strain.

[0066] The SADS-CoV-CH / GD-01 / 2017 strain is a virulent strain isolated from southern China in 2017. According to the disclosure of Y.-L. Yang et al. in Virology 536 (2019) 110-118, the infectious clone of SADS-CoV-CH / GD-01 / 2017 can be rescued. The genome sequence of the infectious clone of SADS-CoV-CH / GD-01 / 2017 can be obtained from GenBank with accession number MK977618.

[0067] An exemplary NSP2 protein of PEDV has an amino acid sequence as set forth in SEQ ID NO:1 or a complete coding sequence as set forth in SEQ ID NO:2. An exemplary NSP2 protein of SADS-CoV has an amino acid sequence as set forth in SEQ ID NO:3 or a complete coding sequence as set forth in SEQ ID NO:8. An exemplary NSP2 protein of TGEV has an amino acid sequence as set forth in SEQ ID NO:4 or a complete coding sequence as set forth in SEQ ID NO:9. An exemplary NSP2 protein of PRCV has an amino acid sequence as set forth in SEQ ID NO:5 or a complete coding sequence as set forth in SEQ ID NO:10. An exemplary NSP2 protein of PHEV has an amino acid sequence as set forth in SEQ ID NO:6 or a complete coding sequence as set forth in SEQ ID NO:11. An exemplary NSP2 protein of PDCoV has an amino acid sequence as set forth in SEQ ID NO:7 or a complete coding sequence as set forth in SEQ ID NO:12.

[0068] In some embodiments, the NSP2 protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 1 and 3-7.

[0069] In some embodiments, the complete NSP2 coding sequence has a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 2 and 8-12.

[0070] "Sequence identity" between two polypeptide sequences refers to the percentage of identical amino acids between the sequences. Methods for evaluating the level of sequence identity between amino acid or nucleotide sequences are known in the art. For example, sequence analysis software is commonly used to determine the identity of amino acid sequences. For example, identity can be determined by using the BLAST program of the NCBI database. For determination of sequence identity, see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0071] As used herein, it is particularly to be understood that the term "sequence identity to the sequence of SEQ ID NO: X" is equivalent to the term "having sequence identity to the sequence of SEQ ID NO: X over the length of SEQ ID NO: X" or the term "having sequence identity to the sequence of SEQ ID NO: X over the entire length of SEQ ID NO: X", respectively. In the present context, "X" is any integer selected from 1 to 32, such that "SEQ ID NO: X" represents any SEQ ID NO mentioned herein.

[0072] In one aspect, the present invention provides a recombinant porcine coronavirus, in particular a recombinant porcine coronavirus as described above, which is

[0073] i) the recombinant PEDV deposited with the CCTCC under accession number CCTCC V202263 on August 10, 2022, or any of its progeny or descendants; or

[0074] ii) a recombinant PEDV, whose genomic sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity with the recombinant PEDV deposited with the CCTCC under the accession number CCTCC V202263 on August 10, 2022.

[0075] In one aspect, the invention provides an isolated nucleic acid encoding a recombinant porcine coronavirus of the invention.

[0076] The term "isolated" as used herein in the context of nucleic acids refers in particular to separation from its production environment and preferably to processing by one or more purification steps which separate the nucleic acid from other molecules with which it has been associated during its production.

[0077] The term "nucleic acid" refers to a polynucleotide, including a DNA molecule, an RNA molecule, a cDNA molecule or a derivative. The term includes single-stranded and double-stranded polynucleotides. The nucleic acids of the present invention include isolated polynucleotides (i.e., isolated from their natural environment) and genetically modified forms. In addition, chemically modified polynucleotides are also included, including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificially modified polynucleotides such as biotinylated polynucleotides. In addition, it should be understood that due to the degeneracy of the genetic code, the recombinant porcine coronavirus of the present invention can be encoded by a large number of polynucleotides.

[0078] In one aspect, the invention provides a vector comprising a nucleic acid of the invention.

[0079] The term "vector" includes bacteriophage, plasmid, virus or retroviral vector and artificial chromosome, such as bacterial or yeast artificial chromosome. In addition, the term also relates to a targeting construct, which allows the targeting construct to be randomly or site-specifically integrated into genomic DNA. Such a target construct preferably includes DNA of sufficient length for homologous or heterologous recombination described in detail below. The vector including the nucleic acid of the present invention preferably further includes a selection marker for propagation and / or selection in a host. The vector can be incorporated into a host cell by various techniques well known in the art. For example, a plasmid vector can be introduced into a precipitate such as a calcium phosphate precipitate or a rubidium chloride precipitate, or into a complex with a charged lipid, or into a carbon-based cluster such as fullerene. Alternatively, a plasmid vector can be introduced by heat shock or electroporation techniques. If the vector is a virus, before being applied to a host cell, a suitable packaging cell system can be used for in vitro packaging. Retroviral vectors can be replication-competent or replication-defective. In the latter case, viral propagation usually only occurs in a complementary host / cell. More preferably, the polynucleotide is operably connected to an expression control sequence that allows expression in a prokaryotic or eukaryotic cell or its separated fraction. The expression of the polynucleotide includes transcription of the polynucleotide, preferably transcribed as translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known in the art. They preferably include regulatory sequences that ensure transcription initiation and optionally poly-A signals that ensure transcription termination and transcript stability. Other regulatory elements may include transcription and translation enhancers. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, lac, trp or tac promoters in Escherichia coli (E.coli), and examples of regulatory elements that allow expression in eukaryotic host cells are CMV-, SV40-, RSV-promoters (Rous sarcoma virus), CMV-enhancers, SV40-enhancers or globin introns in yeast or mammals and other animal cells. In addition, inducible expression control sequences may be used in expression vectors included in the present invention. Such inducible vectors may include tet or lac operator sequences or sequences that can be induced by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. For example, these techniques are described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).

[0080] In one aspect, the present invention provides an immunogenic composition comprising the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention, or the vector of the present invention.

[0081] The term "immunogenic composition" refers to a composition comprising at least one antigen that elicits an immune response in a host to which the immunogenic composition is administered. This immune response may be a cellular and / or antibody-mediated immune response to the immunogenic composition of the invention. Preferably, the immunogenic composition induces an immune response, more preferably confers protective immunity against one or more clinical symptoms of porcine coronavirus infection.

[0082] Porcine coronavirus in the context of the present invention is most preferably PEDV. In this respect, reference herein to "porcine coronavirus infection" or "porcine coronavirus infection", respectively, is preferably to be understood as "PEDV infection" or "PEDV infection", respectively.

[0083] "Antigen" as used herein refers to, but is not limited to, a component that induces an immune response to an immunogenic composition or vaccine of interest including the antigen or its immunologically active component in a host.Antigens or immunologically active components can be complete microorganisms (in an inactivated or modified live form), or any fragment or part thereof, which can induce an immune response in a host if administered to a host.Antigens can be or can include complete living organisms, either in their original form, or attenuated organisms in so-called modified live vaccines (MLV).Antigens can also include appropriate elements of an organism (subunit vaccines), wherein these elements are produced by destroying the entire organism or the growth culture of these organisms and subsequent purification steps to produce the desired structure, or by appropriately manipulating a suitable system such as, but not limited to, a synthetic process induced by bacteria, insects, mammals or other species, and optionally by subsequent separation and purification steps, or by inducing a synthetic process in an animal in need of a vaccine by using a suitable pharmaceutical composition directly incorporated into genetic material (polynucleotide vaccination).Antigens can include complete organisms inactivated by appropriate methods in so-called inactivated vaccines (KV).

[0084] Typically, an "immune response" includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells and / or cytotoxic T cells and / or gamma-delta T cells, specifically directed against one or more antigens included in the immunogenic composition of the invention. Preferably, the host will display a protective immune response or a therapeutic response.

[0085] A "protective immune response" or "protective immunity" will be demonstrated by a reduction or absence of clinical symptoms normally displayed by an infected host, a faster recovery time and / or a lower duration of infectivity or lower titers of pathogens in the tissues or body fluids or excretions of the infected host.

[0086] In some embodiments, the immunogenic compositions of the invention further include a pharmaceutically acceptable carrier.

[0087] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, adjuvants, immunostimulants, and combinations thereof.

[0088] "Diluents" may include water, saline, glucose, ethanol, glycerol, etc. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, lactose, etc. Stabilizers include albumin and alkali metal salts of ethylenediaminetetraacetic acid, etc.

[0089] An immunogenic composition is described as a "vaccine" where the host exhibits a protective immune response resulting in increased resistance to new infection and / or reduced clinical severity of disease.

[0090] In some embodiments, the immunogenic composition of the invention is a vaccine. In some embodiments, the vaccine further comprises an adjuvant.

[0091] "Adjuvants" as used herein may include aluminum hydroxide and aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions. The emulsions may be based in particular on light liquid paraffin oil (Ph. Eur. type); isoprenoid oils such as squalane or squalene; oils resulting from the oligomerization of olefins, in particular isobutylene or decene; esters of acids or alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glycerol tri-(caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearates. The oil is used in combination with an emulsifier to form an emulsion. Emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, esters of mannide (e.g., anhydromannitol oleate), esters of glycols, esters of polyglycerols, esters of propylene glycol and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular Pluronic products. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, DES), John Wiley and Sons, NY, pp51 to 94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

[0092] In one aspect, the present invention provides an immunogenic composition of the present invention for use in

[0093] i) Immunization of pigs;

[0094] ii) eliciting an immune response against porcine coronavirus in pigs;

[0095] iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or

[0096] iv) Reducing mortality caused by porcine coronavirus infection in pigs.

[0097] In some embodiments, the porcine coronavirus is PEDV and the porcine coronavirus infection is a PEDV infection.

[0098] In some embodiments, the porcine coronavirus is SADS-CoV and the porcine coronavirus infection is a SADS-CoV infection.

[0099] In some embodiments, the pig is a piglet or a sow.

[0100] In one aspect, the present invention provides an immunogenic composition of the present invention for use in

[0101] i) reducing or preventing clinical symptoms or disease caused by PEDV infection in piglets that are to be suckled by sows to which the immunogenic composition of the invention has been administered; and / or

[0102] ii) reducing mortality caused by PEDV infection in piglets which are suckled by sows to which the immunogenic composition of the invention has been administered.

[0103] In one aspect, the present invention provides a use of the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention or the vector of the present invention in preparing an immunogenic composition, wherein the immunogenic composition is used for

[0104] i) Immunization of pigs;

[0105] ii) eliciting an immune response against porcine coronavirus in pigs;

[0106] iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or

[0107] iv) Reducing mortality caused by porcine coronavirus infection in pigs.

[0108] In some embodiments, the porcine coronavirus is PEDV and the porcine coronavirus infection is a PEDV infection.

[0109] In some embodiments, the porcine coronavirus is SADS-CoV and the porcine coronavirus infection is SADS-CoV infection. In some embodiments, the pig is a piglet or a sow.

[0110] In some embodiments, the immunogenic composition further includes a pharmaceutically acceptable carrier.

[0111] In some embodiments, the immunogenic composition is a vaccine.

[0112] In some embodiments, the vaccine also includes an adjuvant.

[0113] In one aspect, the present invention provides a use of the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention or the vector of the present invention in preparing an immunogenic composition, wherein the immunogenic composition is used for

[0114] i) reducing or preventing clinical symptoms or disease caused by PEDV infection in piglets that are to be suckled by sows to which the immunogenic composition has been administered; and / or

[0115] ii) reducing mortality caused by PEDV infection in piglets suckled by sows to which the immunogenic composition has been administered.

[0116] In some embodiments, the immunogenic composition further includes a pharmaceutically acceptable carrier.

[0117] In some embodiments, the immunogenic composition is a vaccine.

[0118] In some embodiments, the vaccine also includes an adjuvant.

[0119] In one aspect, the present invention provides a method for

[0120] i) Immunization of pigs;

[0121] ii) eliciting an immune response against porcine coronavirus in pigs;

[0122] iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or

[0123] iv) reducing mortality caused by porcine coronavirus infection in pigs,

[0124] The method comprises administering to a pig an effective amount of the immunogenic composition of the invention.

[0125] In some embodiments, the porcine coronavirus is PEDV and the porcine coronavirus infection is a PEDV infection.

[0126] In some embodiments, the porcine coronavirus is SADS-CoV and the porcine coronavirus infection is a SADS-CoV infection.

[0127] In some embodiments, the pig is a piglet or a sow.

[0128] In one aspect, the invention provides a method for i) reducing or preventing clinical symptoms or disease caused by PEDV infection in piglets, and / or ii) reducing mortality caused by PEDV infection in piglets, wherein the piglets are suckled by sows to which an immunogenic composition of the invention has been administered.

[0129] In one aspect, the present invention provides a method for ii) reducing clinical symptoms or disease caused by PEDV infection in piglets and / or ii) reducing mortality caused by PEDV infection in piglets, wherein the method comprises

[0130] - Administering the immunogenic composition of the invention to sows

[0131] - Allowing piglets to be suckled by sows, or respectively allowing piglets to suckle sows.

[0132] Administration of the immunogenic composition of the present invention results in the production of PEDV-specific antibodies in sows. Maternally derived antibodies from the mother of the sow are passively transferred to newborn piglets through colostrum and / or milk, thereby providing protection for newborn piglets against PEDV infection.

[0133] The term "immunization" relates to active immunization by administering an immunogenic composition to a subject to be immunized, thereby eliciting an immune response against the antigens included in this immunogenic composition.

[0134] Preferably, the immunization results in a reduced incidence of infection with a particular porcine coronavirus, such as PEDV, in a herd or a reduced severity of clinical symptoms caused by or associated with infection with a particular porcine coronavirus, such as PEDV.

[0135] In addition, immunization of a subject in need with the immunogenic composition provided herein results in preventing the subject from being infected with a porcine coronavirus, such as PEDV infection. Even more preferably, immunization results in an effective, durable immune response against porcine coronavirus infection, such as PEDV infection. It should be understood that the time period will last for more than 1 month, preferably more than 2 months, preferably more than 3 months, more preferably more than 4 months, more preferably more than 5 months, and more preferably more than 6 months. It should be understood that immunization may not be effective in all immunized subjects. However, the term requires that a majority of the subjects in the herd are effectively immunized.

[0136] Preferably, a population of subjects is contemplated herein, which normally (i.e., without immunization) would develop clinical symptoms typically caused by or associated with porcine coronavirus infection, such as PEDV infection. It will be apparent to one skilled in the art that a population of subjects is effectively immunized. Preferably, immunization should be effective if the incidence or severity of clinical symptoms is reduced by at least 10%, more preferably at least 20%, still more preferably at least 30%, even more preferably at least 40%, still more preferably at least 50%, even more preferably at least 60%, still more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90%, still more preferably at least 95% and most preferably at 100% in at least 33%, at least 50%, at least 60%, at least 70%, at least 80%, ...

[0137] The term "treatment and / or prevention" refers to reducing the incidence of a specific porcine coronavirus, such as PEDV infection in a herd or reducing the severity of clinical symptoms caused by or associated with a specific porcine coronavirus, such as PEDV infection. Therefore, the term "treatment and / or prevention" also refers to reducing the number of subjects infected with a specific porcine coronavirus, such as PEDV (= a reduction in the incidence of a specific porcine coronavirus, such as PEDV infection) in a herd or reducing the severity of clinical symptoms typically associated with or caused by a porcine coronavirus, such as PEDV infection, or reducing viral shedding after infection with a specific porcine coronavirus, such as PEDV, or preventing or reducing diarrhea after infection with a specific porcine coronavirus, such as PEDV, in a group of subjects that have received an effective amount of an immunogenic composition as provided herein, compared to a group of subjects that have not received such an immunogenic composition.

[0138] "Treatment and / or prevention" generally involves administering an effective amount of the immunogenic composition of the present invention to a subject or group of subjects in need of such treatment / prevention or who may benefit from such treatment / prevention. The term "treatment" refers to the administration of an effective amount of the immunogenic composition once the subject or at least some of the subjects in the herd have been infected with such a porcine coronavirus such as PEDV and wherein such subjects have shown some clinical symptoms caused by or associated with such a porcine coronavirus such as PEDV infection. The term "prevention" refers to administration to a subject before the subject is infected with a porcine coronavirus such as PEDV, or at least when no subject in the subject or a group of subjects does not show clinical symptoms caused by or associated with such a porcine coronavirus such as PEDV. The terms "prevention" and "preventing" are used interchangeably in this application.

[0139] The term "effective amount" as used herein refers to, but is not limited to, the amount of an antigen that elicits or is capable of eliciting an immune response in a subject. Such an effective amount can reduce the incidence of a specific porcine coronavirus such as PEDV infection in a herd or reduce the severity of clinical symptoms of a specific porcine coronavirus such as PEDV infection.

[0140] Preferably, the incidence or severity of clinical symptoms is reduced by at least 10%, more preferably at least 20%, still more preferably at least 30%, even more preferably at least 40%, still more preferably at least 50%, still more preferably at least 60%, still more preferably at least 70%, still more preferably at least 80%, still more preferably at least 90%, still more preferably at least 95%, and most preferably 100%, compared to subjects who were not treated or treated with an immunogenic composition available prior to the present invention but were subsequently infected with a specific porcine coronavirus such as PEDV.

[0141] As used herein, the term "clinical symptoms" refers to signs of infection of a subject with a porcine coronavirus such as PEDV. Examples of such clinical symptoms include, but are not limited to, viral load, diarrhea, shedding, elevated body temperature, mortality, gross pathological lesions in the intestine, depression, weight loss, reduced growth rate, and decreased appetite. However, clinical symptoms also include, but are not limited to, clinical symptoms directly observed from live animals. Examples of clinical symptoms that can be directly observed from live animals include weight loss, reduced growth rate, decreased appetite, dehydration, watery diarrhea, vomiting, lameness, lethargy, emaciation, and unhappiness, among others.

[0142] Preferably, reduced morbidity or severity of clinical symptoms in treated subjects refers to reduced weight loss, lower viral load, reduced diarrhea, reduced shedding, reduced rectal temperature, mortality, reduced gross pathological lesions in the intestine, or a combination thereof, compared to subjects not treated or treated with an immunogenic composition available prior to the present invention but subsequently infected with a specific porcine coronavirus, such as PEDV.

[0143] The term "reduced mortality" refers to a reduction in mortality of at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%, compared to untreated (non-immunized) subjects who are subsequently infected with a particular porcine coronavirus, such as PEDV.

[0144] Preferably, the subject is a pig, such as a piglet or a sow.

[0145] In one aspect, the present invention provides a method for inducing porcine coronavirus, such as PEDV-specific antibodies in a subject, wherein the method comprises administering the immunogenic composition herein to the subject. Preferably, the subject is a pig, such as a piglet or a sow.

[0146] The term "antibodies specific for porcine coronaviruses such as PEDV" refers to detectable anti-porcine coronaviruses such as anti-PEDV antibodies. In addition, anti-porcine coronavirus antibodies in the subject have been produced in response to vaccination with the present invention. Preferably, the antibody titer is detectable and quantifiable in a specific immunoassay.

[0147] In various aspects of the invention, the immunogenic composition is administered to the pig within the first two months, more preferably within the first month.

[0148] In various aspects of the invention, the immunogenic composition is administered to the pig within the first month of age. Thus, it must be understood that the immunogenic composition may be administered to the pig within, for example, the first three weeks or the first two weeks.

[0149] In various aspects of the invention, the immunogenic composition is administered to sows during pregnancy and lactation.

[0150] In various aspects of the invention, the immunogenic composition is administered to pregnant sows at least twice before farrowing, preferably three times before farrowing, and more preferably twice before farrowing ("repeat dose"). Preferably, pregnant sows are vaccinated twice with the immunogenic composition of the present invention before farrowing, a single dose of the composition. However, when the immunogenic composition is administered to sows twice, the first administration should be performed 12-4 weeks before farrowing, more preferably 9-5 weeks before farrowing. The second administration should be performed 8-1 weeks before farrowing, more preferably 6-1 weeks before farrowing.

[0151] In various aspects of the invention, the immunogenic composition is administered in two or more doses.

[0152] In various aspects of the invention, the immunogenic composition is administered to the sow twice, the first administration between 9-5 weeks before farrowing and the second administration between 6-1 week before farrowing.

[0153] In various aspects of the invention, it is preferred to administer the immunogenic composition locally or systemically. Suitable routes of administration conventionally used are oral or parenteral administration, such as intranasal, intravenous, intramuscular, intraperitoneal, subcutaneous and inhalation. However, depending on the nature and mode of action of the compound, the immunogenic composition may also be administered by other routes.

[0154] In various aspects of the invention, the immunogenic composition is administered intranasally, mucosally, orally, intradermally, or intramuscularly.

[0155] In various aspects of the invention, the immunogenic composition comprises 1×10 2 Up to 1×10 9 TCID 50 / ml, more preferably 1×10 3 Up to 1×10 7 TCID 50 / ml, most preferably 1×10 4 Up to 1×10 6 TCID 50 / ml of the recombinant porcine coronavirus of the present invention.

[0156] In various aspects of the invention, the immunogenic composition comprises 1×10 3 Up to 1×10 7 TCID 50 / ml of the recombinant porcine coronavirus of the present invention.

[0157] The term "TCID 50 " / ml" refers to the measure of infectious virus titer. Specifically, tissue culture infectious dose fifty per milliliter (TCID50 / ml) gives the dilution of the virus preparation at which 50% of a number of cell cultures inoculated in parallel with the dilution are infected.

[0158] In one aspect, the present invention provides a method for attenuating porcine coronavirus, comprising deleting the entire nonstructural protein 2 (NSP2) coding sequence from its genome.

[0159] In some embodiments, the method comprises deleting the entire NSP2 gene from its genome.

[0160] In some embodiments, the methods produce an attenuated virus capable of infecting a host cell.

[0161] In some embodiments, the porcine coronavirus is an alphacoronavirus, a betacoronavirus, or a deltacoronavirus.

[0162] In some embodiments, the porcine coronavirus is selected from porcine epidemic diarrhea virus (PEDV), porcine acute diarrhea syndrome-coronavirus (SADS-CoV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV), and porcine delta coronavirus (PDCoV).

[0163] In some embodiments, the porcine coronavirus is PEDV.

[0164] In some embodiments, the porcine coronavirus is genotype 2 PEDV.

[0165] In some embodiments, the porcine coronavirus is PEDV strain PEDV-ZJU / G2 / 2013.

[0166] In some embodiments, the porcine coronavirus is SADS-CoV.

[0167] In some embodiments, the porcine coronavirus is SADS-CoV strain SADS-CoV-CH / GD-01 / 2017.

[0168] In some embodiments, the NSP2 protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 1 and 3-7.

[0169] In some embodiments, the complete NSP2 coding sequence has a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 2 and 8-12.

[0170] In one aspect, the present invention provides a method for producing a recombinant porcine coronavirus of the present invention, comprising

[0171] introducing the isolated nucleic acid of the invention or the vector of the invention into a host cell, and

[0172] Recovering the recombinant porcine coronavirus produced by the host cells.

[0173] In some embodiments, the host cell is a eukaryotic cell line.In some embodiments, the host cell is a Vero cell, a ST cell, a PK cell (eg, a PK15 cell) or a BHK cell.

[0174] In one aspect, the invention provides a method for distinguishing an animal infected with a porcine coronavirus from an animal vaccinated with an immunogenic composition of the invention, comprising:

[0175] a) obtaining a sample, and

[0176] b) Testing the sample in immunoassays and / or genomic profiling assays.

[0177] In some embodiments, the method includes determining the presence of a NSP2 coding sequence, a NSP2 protein, or an antibody against a NSP2 protein in a sample.

[0178] The term "sample" refers to a body fluid sample, a separated cell sample or a sample from a tissue or organ. The body fluid sample can be obtained by well-known techniques, preferably including blood, plasma, serum or urine samples, more preferably blood, plasma or serum samples. The tissue or organ sample can be obtained from any tissue or organ by, for example, biopsy. The separated cells can be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting.

[0179] The following clauses are also described herein and are part of the present disclosure:

[0180] Item 1. A recombinant porcine coronavirus comprising a deletion of the entire nonstructural protein 2 (NSP2) coding sequence in its genome.

[0181] Item 2. The recombinant porcine coronavirus of Item 1, wherein the deletion of the entire nonstructural protein 2 (NSP2) coding sequence is a complete deletion of the NSP2 gene.

[0182] Item 3. The recombinant porcine coronavirus of Item 1 or 2, wherein the virus is capable of infecting a host cell.

[0183] Item 4. The recombinant porcine coronavirus of any one of items 1 to 3, wherein the recombinant porcine coronavirus is attenuated.

[0184] Clause 5. The recombinant porcine coronavirus of any one of clauses 1 to 4, wherein the porcine coronavirus is an alphacoronavirus, a betacoronavirus or a deltacoronavirus.

[0185] Item 6. The recombinant porcine coronavirus of Item 5, wherein the porcine coronavirus is selected from porcine epidemic diarrhea virus (PEDV), porcine acute diarrhea syndrome-coronavirus (SADS-CoV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine delta coronavirus (PDCoV).

[0186] Item 7. The recombinant porcine coronavirus of Item 6, wherein the porcine coronavirus is PEDV.

[0187] Item 8. The recombinant porcine coronavirus of Item 7, wherein the porcine coronavirus is genotype 2 PEDV.

[0188] Item 9. The recombinant porcine coronavirus of Item 8, wherein the recombinant porcine coronavirus is derived from the PEDV-ZJU / G2 / 2013 strain.

[0189] Item 10. The recombinant porcine coronavirus of Item 6, wherein the porcine coronavirus is SADS-CoV.

[0190] Item 11. The recombinant porcine coronavirus of Item 8, wherein the recombinant porcine coronavirus is derived from the SADS-CoV-CH / GD-01 / 2017 strain.

[0191] Clause 12. The recombinant porcine coronavirus of any of clauses 1 to 11, wherein the NSP2 protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to any of SEQ ID NOs: 1 and 3-7.

[0192] Clause 13. The recombinant porcine coronavirus of any one of clauses 1 to 12, wherein the complete NSP2 coding sequence has a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to any one of SEQ ID NOs: 2 and 8-12.

[0193] Clause 14. A recombinant porcine coronavirus, in particular the recombinant porcine coronavirus of any one of clauses 1 to 9, which is

[0194] i) the recombinant PEDV deposited with the CCTCC under accession number CCTCC V202263 on August 10, 2022, or any of its progeny or descendants; or

[0195] ii) a recombinant PEDV, whose genomic sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity with the recombinant PEDV deposited with the CCTCC under the accession number CCTCC V202263 on August 10, 2022.

[0196] Item 15. An isolated nucleic acid encoding a recombinant porcine coronavirus according to any one of items 1 to 14.

[0197] Item 16. A vector comprising the nucleic acid of Item 15.

[0198] Clause 17. An immunogenic composition comprising a recombinant porcine coronavirus according to any one of clauses 1 to 14, an isolated nucleic acid according to clause 15 or a vector according to clause 16.

[0199] Clause 18. The immunogenic composition of Clause 17, further comprising a pharmaceutically acceptable carrier.

[0200] Clause 19. The immunogenic composition of clause 17 or 18, wherein the immunogenic composition is a vaccine.

[0201] Clause 20. The immunogenic composition of Clause 19, wherein the vaccine further comprises an adjuvant.

[0202] Clause 21. The immunogenic composition of any one of clauses 17 to 20, for use in

[0203] i) Immunization of pigs;

[0204] ii) eliciting an immune response against porcine coronavirus in pigs;

[0205] iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or

[0206] iv) Reducing mortality caused by porcine coronavirus infection in pigs.

[0207] Clause 22. The immunogenic composition for use according to Clause 21, wherein the porcine coronavirus is PEDV, and / or the porcine coronavirus infection is a PEDV infection.

[0208] Clause 23. An immunogenic composition for use according to Clause 21, wherein the porcine coronavirus is SADS-CoV, and / or the porcine coronavirus infection is a SADS-CoV infection.

[0209] Clause 24. The immunogenic composition for use according to any one of clauses 21 to 23, wherein the pig is a piglet or a sow.

[0210] Clause 25. The immunogenic composition of any one of clauses 17 to 20, for use

[0211] i) reducing or preventing clinical symptoms or disease caused by PEDV infection in piglets which are to be suckled by sows to which the immunogenic composition of any one of clauses 15 to 18 has been administered; and / or

[0212] ii) reducing mortality caused by PEDV infection in piglets which are to be suckled by sows to which the immunogenic composition of any one of clauses 17 to 20 has been administered.

[0213] Clause 26. Use of the recombinant porcine coronavirus of any one of clauses 1 to 14, the isolated nucleic acid according to clause 15 or the vector according to clause 16 in the preparation of an immunogenic composition for use in

[0214] i) Immunization of pigs;

[0215] ii) eliciting an immune response against porcine coronavirus in pigs;

[0216] iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or

[0217] iv) Reducing mortality caused by porcine coronavirus infection in pigs.

[0218] Clause 27. The use according to Clause 26, wherein the porcine coronavirus is PEDV, and / or the porcine coronavirus infection is PEDV infection.

[0219] Clause 28. The use of Clause 26, wherein the porcine coronavirus is SADS-CoV, and / or the porcine coronavirus infection is SADS-CoV infection.

[0220] Clause 29. The use according to any one of clauses 26 to 28, wherein the pig is a piglet or a sow.

[0221] Clause 30. Use of the recombinant porcine coronavirus of any one of clauses 1 to 14, the isolated nucleic acid according to clause 15 or the vector according to clause 16 in the preparation of an immunogenic composition for use in

[0222] i) reducing or preventing clinical symptoms or disease caused by PEDV infection in piglets that are to be suckled by sows to which the immunogenic composition has been administered; and / or

[0223] ii) reducing mortality caused by PEDV infection in piglets suckled by sows to which the immunogenic composition has been administered.

[0224] Clause 31. The use of any one of Clauses 26 to 30, wherein the immunogenic composition further comprises a pharmaceutically acceptable carrier.

[0225] Clause 32. The use of any one of clauses 26 to 31, wherein the immunogenic composition is a vaccine.

[0226] The use of clause 33.32, wherein the vaccine further comprises an adjuvant.

[0227] Clause 34. A method for

[0228] i) Immunization of pigs;

[0229] ii) eliciting an immune response against porcine coronavirus in pigs;

[0230] iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or

[0231] iv) reducing mortality caused by porcine coronavirus infection in pigs,

[0232] Wherein the method comprises administering to a pig the immunogenic composition of any one of clauses 17-20.

[0233] Clause 35. The method of Clause 34, wherein the porcine coronavirus is PEDV and the porcine coronavirus infection is a PEDV infection.

[0234] Clause 36. The method of Clause 34, wherein the porcine coronavirus is SADS-CoV, and / or the porcine coronavirus infection is SADS-CoV infection.

[0235] Clause 37. The method of any one of clauses 34 to 36, wherein the pig is a piglet or a sow.

[0236] Clause 38. A method for i) reducing or preventing clinical symptoms or disease caused by PEDV infection in piglets, and / or ii) reducing mortality caused by PEDV infection in piglets, wherein the piglets are suckled by sows to which the immunogenic composition of any one of Clauses 17 to 20 has been administered.

[0237] Item 39. A method for attenuating a porcine coronavirus comprising deleting the entire nonstructural protein 2 (NSP2) coding sequence from its genome.

[0238] Clause 40. The method of Clause 39, comprising deleting the entire NSP2 gene from its genome.

[0239] Item 41. The method of Item 39 or 40, which produces an attenuated virus capable of infecting a host cell.

[0240] Clause 42. The method of any one of clauses 39 to 41, wherein the porcine coronavirus is an alphacoronavirus, a betacoronavirus or a deltacoronavirus.

[0241] Item 43. The method of Item 42, wherein the porcine coronavirus is selected from porcine epidemic diarrhea virus (PEDV), porcine acute diarrhea syndrome-coronavirus (SADS-CoV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine delta coronavirus (PDCoV).

[0242] Clause 44. The method of Clause 43, wherein the porcine coronavirus is PEDV.

[0243] Clause 45. The method of clause 44, wherein the porcine coronavirus is genotype 2 PEDV.

[0244] Clause 46. The method of clause 45, wherein the porcine coronavirus is PEDV strain PEDV-ZJU / G2 / 2013.

[0245] Clause 47. The method of Clause 43, wherein the porcine coronavirus is SADS-CoV.

[0246] Clause 48. The method of clause 47, wherein the porcine coronavirus is SADS-CoV strain SADS-CoV-CH / GD-01 / 2017.

[0247] Clause 49. The method of any one of clauses 39 to 48, wherein the NSP2 protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to any one of SEQ ID NOs: 1 and 3-7.

[0248] Clause 50. The method of any one of clauses 39 to 49, wherein the complete NSP2 coding sequence has a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to any one of SEQ ID NOs: 2 and 8-12.

[0249] Clause 51. A method for producing a recombinant porcine coronavirus according to any one of clauses 1 to 14, comprising introducing the isolated nucleic acid according to clause 15 or the vector according to clause 16 into a host cell, and

[0250] Recovering the recombinant porcine coronavirus produced by the host cells.

[0251] Item 52. The method of Item 51, wherein the host cell is a Vero cell, a ST cell or a PK cell.

[0252] Clause 53. A method of distinguishing an animal infected with porcine coronavirus from an animal vaccinated with the immunogenic composition of any one of clauses 17 to 20, comprising

[0253] a) obtaining a sample, and

[0254] b) Testing the sample in immunoassays and / or genomic profiling assays.

[0255] Clause 54. The method of clause 53, comprising determining the presence of NSP2 coding sequences, NSP2 proteins or antibodies against NSP2 proteins in the sample.

[0256] Example

[0257] The following examples further illustrate the present invention by way of illustration. It should be understood that the present invention is not limited to any one of those embodiments described below. Those skilled in the art will appreciate that, in view of the general description of the present invention, the performance, results and findings of these embodiments may be modified and applied in a broader sense.

[0258] Example 1. Generation of rPEDV-delNsp2

[0259] The strategy for rescuing the PEDV ZJU / G2 / 2013 strain with complete deletion of NSP2 coding sequence is as follows Figure 1 shown.

[0260] 1. Amplification of the target fragment of the full-length infectious clone of PEDV-G2-ZJU-ΔNsp2

[0261] RNA was extracted from the supernatant sample of cells infected with the G2 genotype PEDV ZJU / G2 / 2013 strain (a Zhejiang virulent strain isolated by the inventors in 2013) by the Trizol method, and reverse transcribed to obtain full-length cDNA as a template for amplification.

[0262] According to the genome sequence of ZJU / G2 / 2013 strain (accession number: KU558701) in GenBank, 9 pairs of primers were designed using Primerpremier 5.0 software (primer sequences are shown in Table 1) to amplify 9 truncated fragments covering the full length of the PEDV genome except the NSP2 coding sequence. The primers were synthesized by Shangya Biotechnology Co., Ltd.

[0263] The amino acid sequence of the NSP2 protein of PEDV ZJU / G2 / 2013 strain is shown in SEQ ID NO:1.

[0264] The NSP2 protein of PEDV ZJU / G2 / 2013 strain is encoded by the nucleotide sequence shown in SEQ ID NO:2.

[0265] PCR amplification was performed using the 9 pairs of primers in Table 1 and the full-length cDNA of PEDV as a template. The polymerase used was Q5 high-fidelity DNA polymerase purchased from NEB. The products obtained by PCR were subjected to 1% agarose gel electrophoresis, and each fragment was consistent with the expected size. The agarose gel containing the target DNA band was cut out, the target fragment was recovered by a kit, and the DNA concentration was determined. The gel recovery kit was purchased from AxyGen.

[0266] Table 1

[0267]

[0268] 2. Assembly of the full-length infectious clone of PEDV-G2-ZJU-ΔNsp2

[0269] GeneArt Immunoassay (GeneArt Immunoassay, Inc.) was used according to the manufacturer's instructions. TM GeneArt TM High-Order Genetic Assembly System), assemble the fragments by seamless cloning. The 9 cloned DNA fragments were connected to the linearized pSB2μ vector (Yong-Le Yang, et al. Characterization of novel bat-HKU2-like swine enteric alphacoronavirus (SeACoV) infection incultured cells and development of a SeACoV infectious clone. Virology. 2019, 536: 110-118) to obtain the PEDV-G2-ZJU-ΔNsp2 infectious clone recombinant vector. The product was transformed into DH10B competent cells.

[0270] 3. Verification of the full-length infectious clone of PEDV-G2-ZJU-ΔNsp2

[0271] Pick a single clone colony, inoculate it in 5mL liquid LB medium containing chloramphenicol (40μg / mL) resistance, incubate at 37°C for 16 hours, and then extract the plasmid according to the instructions of AxyPrep Plasmid DNA Mini Kit. Obtain the PEDV-G2-ZJU-ΔNsp2 infectious clone recombinant plasmid. For subsequent PCR amplification and verification on the infectious clone, the full-length genome sequence was divided into 26 fragments, each about 1400bp, for sequencing. The sequencing results confirmed that the correct PEDV-G2-ZJU-ΔNsp2 full-length infectious clone plasmid was obtained. The resulting recombinant plasmid was named pSB-PEDV-G2-ZJU-ΔNsp2.

[0272] 4. Virus rescue

[0273] 4.1. Preparation of PEDV-G2-ZJU-ΔNsp2 infectious clone plasmid

[0274] The glycerol bacteria containing the plasmid were inoculated into 1L 2×YT liquid medium containing chloramphenicol (40 μg / mL) resistance at a ratio of 1:100, and cultured at 200 rpm at 37°C for 12 h. The PEDV-G2-ZJU-ΔNsp2 infectious clone plasmid was extracted according to the instructions of the BAC / PAC DNA Isolation Maxi Kit and stored at -20°C for later use.

[0275] 4.2. Construction of pRK5-PEDV-N plasmid

[0276] Using PEDV-G2-ZJU plasmid as a template, the fragment PEDV-N (N gene sequence) was amplified and cloned into the linearized pRK5 plasmid through EcoR I and Xba I double restriction reaction to obtain pRK5-PEDV-N plasmid.

[0277] 4.3. Transfection of PEDV-G2-ZJU-ΔNsp2 plasmid to rescue virus

[0278] according to Figure 2 Rescue of infectious virus was performed. Specifically, BHK-21 cells were plated in 12-well plates, placed in a 5% CO2, 37°C incubator, and transfected when the cell density reached about 75%. According to the instructions of the 3000 transfection kit, the medium was discarded, the cells were washed twice with Opti-MEM, and 1.5 μg of PEDV-G2-ZJU-ΔNsp2 infectious clone plasmid and 1 μg of pRK5-PEDV-N plasmid were added to each well. The medium was changed 6 hours after transfection and observed every day. When cytopathic effects appeared, the rescued virus supernatant was collected by repeated freezing and thawing and named rPEDV-G2-ZJU-ΔNsp2, abbreviated as rPEDV-ΔNsp2 or rPEDV-delNsp2.

[0279] Vero cells were seeded in 12-well plates and placed in a 5% CO2, 37°C incubator and infected when the cell density reached about 90%. Three days after infection, the rescued virus was detected by indirect immunofluorescence (IFA) using anti-PEDV-Nsp2 rabbit polyclonal antibody. PEDV-S antibody was used as a positive control. The results are shown in Figure 3 shown.

[0280] RNA was extracted from cells infected with rPEDV-delNsp2 (48 h) by Trizol method and reverse transcribed into cDNA. The cDNA was used as a template for PCR amplification and identified by agarose gel electrophoresis. Figure 4 RT-PCR characterization of the rescued virus rPEDV-delNsp2 is shown.

[0281] The rescued porcine epidemic diarrhea virus rPEDV-G2-ZJU-ΔNsp2, also known as porcine epidemic diarrhea virus rPEDV-ΔNsp2 or porcine epidemic diarrhea virus rPEDV-delNsp2, was deposited in CCTCC (China Center for Type Culture Collection, CHINA CENTER FOR TYPE CULTURE COLLECTION) on August 10, 2022 under the accession number CCTCCV202263, Wuhan University, Wuhan 430072, People's Republic of China).

[0282] 5. Virus growth characteristics of rPEDV-delNsp2 compared with the parental virus WT-PEDV-G2-ZJU

[0283] 5.1. Plaque assay of rPEDV-G2-ZJU-ΔNsp2

[0284] Vero cells were placed in a 6-well plate. After the cell density reached 95%, the cells were infected with rPEDV-G2-ZJU-ΔNsp2. After 2 hours of infection, the supernatant was discarded and preheated 2% low-melting point agarose was added to the 6-well plate. After solidification, it was transferred to a 37°C incubator and inverted for 96 hours. The cells were fixed with 4% paraformaldehyde at room temperature overnight, and then the agar was discarded and stained with crystal violet. After standing at room temperature for 30 minutes, the crystal violet solution was discarded, and the number of plates was observed after washing with water. The experimental procedures for the parental virus WT-PEDV-G2-ZJU were the same as above. The results are shown in Figure 5 As shown, the plaque size induced by the Nsp2-deficient strain after infection of cells was significantly smaller than that of the parental virus-infected group, indicating that the viral replication kinetics of the Nsp2-deficient strain was impaired.

[0285] 5.2. Plotting the growth curve of rPEDV-G2-ZJU-ΔNsp2 virus

[0286] Vero cells were placed in 8 35 mm cell culture dishes, placed in a 5% CO2, 37°C incubator, and infected when the cell density reached about 90%. rPEDV-G2-ZJU-ΔNsp2 virus supernatant was collected from 8 cell culture dishes by repeated freezing and thawing at 2h, 6h, 12h, 18h, 24h, 36h, 48h and 72h after infection. Vero cells were plated in 8 96-well cell culture plates, placed in a 5% CO2, 37°C incubator, and when the cell density reached about 90%, they were infected with the previously collected 2h, 6h, 12h, 24h, 36h, 48h, 72h viruses after 10-fold gradient dilution. The number of lesions was observed after 72h, and the TCID of the virus was calculated. 50 , and the virus growth curve was drawn. The experimental steps of the parent virus WT-PEDV-G2-ZJU were the same as above. The results are shown in Figure 6 As shown. TCID of the recombinant virus strain lacking NSP2 after 18 h of infection 50 Significantly lower than the TCID of parental virus infection 50 , the viral titer of the deletion strain was reduced.

[0287] Quantification of rPEDV-G2-ZJU-ΔNsp2 RNA

[0288] Vero cells were placed in a 12-well plate, and after the cell density reached 95%, the cells were infected with rPEDV-G2-ZJU-ΔNsp2. Supernatants and cells were collected at 2h, 6h, 12h, 18h, 24h, 36h, 48h, and 72h after infection. RNA from the samples was quantified by real-time PCR. The results showed that 24h after virus infection, the amount of RNA synthesis of rPEDV-G2-ZJU-ΔNsp2 was significantly lower than that of the parental virus ( Figure 7 ).

[0289] 5.4. Western blot detection of rPEDV-G2-ZJU-ΔNsp2

[0290] Vero cells were placed in a 12-well cell plate in a 5% CO2, 37°C incubator and infected when the cell density reached about 90%. PEDV-G2-ZJU-ΔNsp2 cell samples were collected for Western blotting experiments at 2h, 6h, 12h, 18h, 24h, 36h, 48h, and 72h after infection. The experimental steps of the parental virus WT-PEDV-G2-ZJU were the same as above. The Western blot results were consistent with the RNA quantification results, that is, the expression of rPEDV-G2-ZJU-ΔNsp2 N protein was significantly lower than that of the WT-PEDV-G2-ZJU infection group 24h after virus infection ( Figure 8 ), indicating that the viral replication ability of the Nsp2-deficient strain is weaker than that of the wild-type strain.

[0291] Example 2. Safety of rPEDV-delNsp2 in pigs

[0292] The aim of this study was to evaluate the safety of different PEDV vaccine candidates administered to PEDV IgA- and IgG-negative piglets 3–5 days of age.

[0293] 1. Study Design

[0294] The research design is shown in Table 2 below.

[0295] Table 2

[0296]

[0297] 18PEDVIgA-negative sows were randomly divided into 4 groups, 4 sows in each group 1-4 and 2 sows in group 5. 3-5 day old piglets from different groups of sows received different VP (10 5.0 TCID 50 / ml, 2ml / piglet, oral (1ml) and intranasal (0.5ml / nostril) per piglet) or MEM (2ml / piglet, oral (1ml) and intranasal (0.5ml / nostril) per piglet). Included piglets were healthy, weighed >0.9kg at farrowing, negative for PEDV genome in fecal samples, and negative for PEDV antibodies (IgA and IgG) in serum before treatment.

[0298] Approximately 2 weeks before farrowing, all sows are vaccinated with FetalGuard LT-C to provide passive maternal immunity to piglets against neonatal diarrhea caused by beta toxin produced by Clostridium perfringens type C and enterotoxigenic strains of Escherichia coli that produce heat-labile toxins or have K88, K99, 987P or F41 adhesion factors.

[0299] On farrowing day (study day (SD) -3 to SD-5), healthy piglets weighing >0.9 kg were included in the study. Colostrum samples were collected from each sow for laboratory testing to monitor IgG and IgA antibody levels.

[0300] On the day of inoculation (SD0), piglets in groups 1, 2, 3, and 4 were inoculated according to Table 2, and piglets in group 5 were inoculated with MEM as a placebo, which served as a negative control.

[0301] After vaccination, all piglets were clinically observed for 14 consecutive days, twice a day, once at about 9 a.m. and once at about 3 p.m., with emphasis on diarrhea and vomiting.

[0302] On SD0 (before inoculation), SD1, SD3, SD7 and SD14, fecal swabs were collected for PEDV genome detection by RT-PCR method.

[0303] On SD0, SD7, and SD14, blood samples were collected for serum production, and serum samples were transferred to the laboratory for detection of PEDV antibodies by the CCIFA method.

[0304] All piglets that died during the clinical period or were euthanized at the humane endpoint or termination of the study were evaluated by gross necropsy for macroscopic lesions.

[0305] The main parameters to be evaluated include morbidity and mortality after inoculation. Mortality: Analyze the mortality of animals that died related to PEDV infection. Morbidity: 1. Analyze the morbidity of any animal that died related to PEDV infection; 2. Analyze the morbidity of animals with a clinical score of 2 for two or more consecutive days.

[0306] Secondary parameters to be evaluated include:

[0307] - Antibody (CCIFA, Cell Culture-Immunofluorescence Assay) levels on SD 0, 7 and 14 from piglet sera.

[0308] - Virus shedding (RT-PCR) results on SD0, SD1, SD3, SD7 and SD14 from piglet fecal samples.

[0309] Clinical scoring system: Piglets were scored for diarrhea where 0 = normal, 1 = semi-liquid (cow-patty stool) with no consistency formed, 2 = water / liquid content.

[0310] 2. Results

[0311] Fig. 9 The morbidity and mortality after vaccination are shown. Clinical scores and survival rates are shown in Fig.10 middle. Fig.11 The results of viral shedding detected by qRT-PCR are shown. Fig.12 Shown are the PEDV antibody levels in piglet sera at 14 dpi detected by CCIFA. Fig.13 Shown are PEDV antibodies in sow sera tested by CCIFA after exposure. Fig.14 Shown are the levels of PEDV antibodies in piglet sera detected by ELISA.

[0312] From the results we can see that:

[0313] When rPEDV-D (nsp2 deletion mutant) was administered to newborn piglets, no PEDV-related morbidity and mortality were observed. The other two candidate PEDVs were unsafe for newborn piglets.

[0314] Very low levels of PEDV RNA were detected in fecal samples from rPEDV-D (nsp2 deletion mutant) piglets at late time points post infection. Seroconversion was also detected in the rPEDV-D group.

[0315] Sows housed horizontally with piglets infected with the PEDV nsp2 deletion mutant did not seroconvert, indicating that the chance of horizontal transmission of the PEDV nsp2 deletion mutant virus from piglets to sows is very low.

[0316] Therefore, rPEDV-D (nsp2 deletion mutant) is safe for newborn piglets.

[0317] Example 3. Efficacy of rPEDV-delNsp2 in pigs

[0318] The objective of this study was to evaluate the efficacy of the rPEDV-delNsp2 candidate (in Example 2 above and hereinafter this candidate is also referred to as "rPEDV-D") on lactational immunity of 3-5 day old piglets against PEDV GII-WT challenge.

[0319] 1. Study Design

[0320] The research design is shown in Table 3 below.

[0321] Table 3

[0322]

[0323]

[0324] Randomization of sows was applied in this study. Pregnant sows with the same expected farrowing date were divided into four groups, with 4 sows in each group from Groups 1 to 3 serving as the vaccinated / challenged group or the challenged control group, and 2 sows in Group 4 serving as the negative control group.

[0325] Sows included in the study were negative for PEDV antibodies and antigens, PPRSV (Ab), CSFV (Ab), and PRV (gE Ab).

[0326] Piglets from sows that were 3-5 days old and weighed more than 0.9 kg on the day of farrowing were included in the study.

[0327] The events in the study are arranged as shown in Table 4 below:

[0328] Table 4

[0329]

[0330] Note: SD stands for study day, which is also the same as the day after sow immunization, named DPI.

[0331] DPC stands for days post-challenge of piglets.

[0332] 2. Evaluation criteria:

[0333] Main parameters:

[0334] Morbidity after challenge in piglets.

[0335] Mortality of piglets after challenge.

[0336] Secondary parameters:

[0337] Antibody (CCIFA) test:

[0338] Sows: Serum samples from sows on SD 0, 14, 21, 28, 35, day of farrowing and DPC 0 (day of challenge). Colostrum on day of farrowing and milk on day of challenge of piglets.

[0339] Piglets: Serum samples from piglets from DPC 0, 7 and 14.

[0340] Viral shedding (RT-qPCR) test:

[0341] Sows: Faecal samples from sows with SD 0, 1, 3, 7, 10, 22, 24, 28 and 31.

[0342] Piglets: Fecal samples from piglets on DPC 0, 3, 7, 10 and 14.

[0343] Clinical Scoring: All piglets were observed for clinical evaluation once daily from DPC 0 to DPC 14. Piglets were scored for diarrhea where 0 = normal, 1 = semi-liquid (cow-patty stool) with no consistency formed, 2 = water / liquid content.

[0344] Morbidity: 1) To summarize the severity of clinical signs, all animals with a clinical score of 2 at both clinical observation time points were counted and piglets were defined as morbidity. 2) Dead animals associated with PEDV infection or PEDV-like lesions characterized by thin and transparent intestinal walls were analyzed as morbidity.

[0345] Mortality: The mortality of animals that died related to PEDV infection was analyzed.

[0346] Piglets in Group 1 showed at least a beneficial effect with respect to one or more of the following parameters: reduced clinical signs, viral shedding, morbidity and mortality compared to piglets in the corresponding non-vaccinated challenged control group (Group 2).

[0347] Example 4. Generation of rSADS-CoV-delNsp2

[0348] Fig.15 A shows the strategy for rescuing the SADS-CoV CH / GD-01 / 2017 strain with a complete deletion of the NSP2 coding sequence.

[0349] 1. Amplification of the target fragment of the full-length infectious clone of SADS-CoV-ΔNsp2

[0350] The supernatant sample of cells infected with the G2 genotype SADS-CoV CH / GD-01 / 2017 strain was used as a template (Zhejiangvirulent strain isolated by the inventor in 2017; Y. Pan et al. VeterinaryMicrobiology 211 (2017) 15–21). RNA was extracted by Trizol method and reverse transcribed to obtain full-length cDNA as an amplification template.

[0351] Primer premier 5.0 software was used to design 15 pairs of primers (Table 5) based on the genome sequence of the CH / GD-01 / 2017 strain in GenBank (Y.-L. Yang, et al. Virology 536 (2019) 110–118; Genbank accession No.: MK977618) to amplify 9 truncated fragments covering the full length of the SADS-CoV genome except the NSP2 coding sequence. The primers were synthesized by Shangya Biotechnology Co., Ltd.

[0352] 15 pairs of primers were used to perform PCR amplification using the full-length cDNA of SADS-CoV as a template. The polymerase used was Q5 high-fidelity DNA polymerase purchased from NEB. The products obtained by PCR were subjected to 1% agarose gel electrophoresis, and each fragment was consistent with the expected size. The agarose gel containing the target DNA band was cut out, the target fragment was recovered by a kit, and the DNA concentration was determined. The gel recovery kit was purchased from AxyGen.

[0353] Table 5

[0354]

[0355] 2. Assembly of the full-length infectious clone of SADS-CoV-CH / GD-01 / 2017-ΔNsp2

[0356] GeneArt purchased from Thermo Scientific, Inc. TM The High-Order Genetic Assembly System was used to assemble the fragments by seamless cloning according to the manufacturer's manual. The 15 cloned DNA fragments were connected to the linearized pSB2μ vector to obtain the SADS-CoV-CH / GD-01 / 2017-ΔNsp2 infectious clone recombinant plasmid. The product was transformed into DH10B competent cells.

[0357] 3. Validation of the full-length infectious clone of SADS-CoV-CH / GD-01 / 2017-ΔNsp2

[0358] A single clone colony was picked and inoculated into 5 mL of liquid LB medium containing chloramphenicol (40 μg / mL) resistance, incubated at 37 ° C for 16 hours, and then the plasmid was extracted according to the instructions of the AxyPrep Plasmid DNA Mini Kit. The SADS-CoV-CH / GD-01 / 2017-ΔNsp2 infectious clone recombinant plasmid was obtained. For subsequent PCR amplification and verification on the infectious clone, the full-length genome sequence was divided into 26 fragments, each about 1400 bp, for sequencing. The sequencing results confirmed that the correct SADS-CoV-CH / GD-01 / 2017-ΔNsp2 full-length infectious clone plasmid was obtained. The resulting recombinant plasmid was named pSB-SADS-CoV-CH / GD-01 / 2017-ΔNsp2.

[0359] 4. Virus rescue

[0360] 4.1. Preparation of the infectious clone plasmid of SADS-CoV-CH / GD-01 / 2017-ΔNsp2

[0361] The glycerol bacteria containing the plasmid were inoculated into 1L 2×YT liquid medium containing chloramphenicol (40 μg / mL) resistance at a ratio of 1:100, and cultured at 37°C and 200 rpm for 12 h. The SADS-CoV-CH / GD-01 / 2017-ΔNsp2 infectious clone plasmid was extracted according to the instructions of the BAC / PAC DNA Isolation Maxi Kit and stored at -20°C for later use.

[0362] 4.2. Construction of pRK5-SADS-CoV-N plasmid

[0363] Using the SADS-CoV-CH / GD-01 / 2017 plasmid as a template, the fragment SADS-CoV-N (N gene sequence) was amplified and cloned into the linearized pRK5 plasmid by EcoR I and Xba I double restriction reaction to obtain the pRK5-SADS-CoV-N plasmid.

[0364] 4.3. Transfection of SADS-CoV-CH / GD-01 / 2017-ΔNsp2 plasmid to rescue virus

[0365] according to Figure 2 Rescue of infectious virus was performed. Specifically, BHK-21 cells were plated in 12-well plates, placed in a 5% CO2, 37°C incubator, and transfected when the cell density reached about 75%. According to the instructions of the 3000 transfection kit, the medium was discarded, the cells were washed twice with Opti-MEM, and 1.5 μg of SADS-CoV-CH / GD-01 / 2017-ΔNsp2 infectious clone plasmid and 1 μg of pRK5-SADS-CoV-N plasmid were added to each well. The medium was changed 6 hours after transfection and observed every day. When cytopathic effects appeared, the rescued virus supernatant was collected by repeated freezing and thawing and named rSADS-CoV-CH / GD-01 / 2017-ΔNsp2, abbreviated as rSADS-CoV-ΔNsp2 or rSADS-CoV-delNsp2.

[0366] Vero cells were seeded in 12-well plates and placed in a 5% CO2, 37°C incubator and infected when the cell density reached about 90%. Three days after infection, the rescued viruses were detected by indirect immunofluorescence (IFA) using anti-SADS-CoV-Nsp2 rabbit polyclonal antibody. The IFA results are shown in Fig.15 As shown in B.

[0367] RNA was extracted from cells infected with rSADS-CoV-delNsp2 (48 h) by the Trizol method and reverse transcribed into cDNA. The cDNA was used as a template for PCR amplification and identified by agarose gel electrophoresis and sequencing. Fig.15 As shown in C, Fig.15 The sequences in C are designated as SEQ ID NOs: 65 and 66.

[0368] 5. Virus growth characteristics of rSADS-CoV-delNsp2 compared with the parental virus WT-SADS-CoV-CH / GD-01 / 2017

[0369] Vero cells were plated in eight 35 mm cell culture dishes in a 5% CO2, 37°C incubator and infected when the cell density reached about 90%. rSADS-CoV-CH / GD-01 / 2017-ΔNsp2 virus supernatants were collected from eight cell culture dishes by repeated freezing and thawing at 2h, 6h, 12h, 18h, 24h, 36h, 48h and 72h after infection. Vero cells were plated in eight 96-well cell culture plates in a 5% CO2, 37°C incubator and infected with previously collected 2h, 6h, 12h, 24h, 36h, 48h, 72h viruses after 10-fold gradient dilution when the cell density reached about 90%. The number of lesions was observed after 72h, the TCID50 of the virus was calculated, and the virus growth curve was plotted. The experimental procedures for the parental virus WT-SADS-CoV-CH / GD-01 / 2017 were the same as above. Growth curve Fig.15 As shown in D.

[0370] Example 5. Safety and efficacy of rPEDV-delNsp2 in pigs

[0371] The safety of the rSADS-CoV-CH / GD-01 / 2017-ΔNsp2 virus was evaluated by administering the virus to pigs, with the wild-type SADS-CoV CH / GD-01 / 2017 strain as a control.

[0372] The efficacy of the rSADS-CoV-CH / GD-01 / 2017-ΔNsp2 virus was evaluated by administering the virus to pigs and then challenging the pigs with wild-type SADS-CoV.

[0373] sequence:

[0374] Some of the sequences mentioned in this article are listed below:

[0375] SEQ ID NO: 1: Amino acid sequence of PEDV NSP2

[0376] NIVPVDQYMCGADGKPVLQESEWEYTDFFADSEDGQLNIAGITYVKAWIVERSDVSYASQNLTSIKSITYCSTYEHTFPDGTAMKVARTPKIKKTVVLSEPLATIYREIGSPFVDNGSDARSIIKRPVFLHAFVKCKCGSYHWTVGDWTSYVSTCCGFKCKPVLVASCSATPGSVVVTRAGAGTGVKYYNNMFLRHVADIDGLAFWRILKVQSKDDLACSGKFLEHHEEGFTDPCYFLNDSSIATKLKFDILSGKFSDEVKQAIFAGHVVVGSALVDIVDDALGQPWFIRKLGDLASAAWEQLKAVVRGLNLLSDEVVLFGKRLSCATLSIVNGVFEFIAEVPGTLAAAVTVFVNFLNELFESACDCLKVGGKTFNKVGSYVLFDNALVKLVKAKVRGPRQAGVCEVRYTSLVIGSTTKVVSKRVENANVNLVVVDEDVTLNTTGRTVVVDGLAFFESDGFYRHLADADVVIEHPVYKSACELKPVFECDPIPDFPMPVAASVAELCVQTDLLLKNYNTPYKTYSCVVRGDKCCITCTLHITAPSYMEDAANFVDLCTKNIGTAGFHEFYITAHEQQDLQGFVTTCCTMSGFECFMPIIPQCPAVLEEIDGGSIWRSFITGLNTMWDFCKHLKVSFGLDGIVVTVARKFKRLGALLAEMYNTYLLTVVENLVLAGVSFKYYATSVPKIVLGCCFHSVKSVLASAFQIPVQAGIEKFKVFLNCVHPVVPRVIETSFVELEETTFKPPALNGSIAIVDGFAFYYDGTLYYPTDGNSVVPICFKKKGG

[0377] SEQ ID NO: 2: The complete coding sequence of PEDV NSP2

[0378]

[0379] SEQ ID NO: 3: Amino acid sequence of SADS-CoV NSP2

[0380] NVVSVDNYMCGADGKPAVPSEQWSFVEHFDDDTDEITVNGVTFRHVWNTVRADDPYEKQGLLSIKSIEYISNVPHKLPNGSVLGVAGTPKKRKAIVLDEKYSKLYDACGVPFVTNGKSISEVVPKPLFLHALVKCKCDNESWTVGDWTGFKTTCCGSTGKVTTLAVGDVTPGDIVFTTLGAGKGTKFFCGLVLTFVDTLEGVSAWRVVKAYTVDKFVASSNFDEHNHVMSLDQCSFDNFSPISVALKFSLLRGNCCDDVKFAVATGVIDIGLGVFDVNDTVFENVPWFVQKFEFLKPAWDALKQAIINLGVTSKAVLSFIKSLCSAAFSVVDGVPVIVCTVAERFSKAFTDFLSYTRECFTALCDDIVVFGVKCKAVGDYIIFNNSVVKTVKAKIKGVKEAGLKTMTYTQCLLGSTKPVKVKRVERSVAQLKVVDTAVPLKQEGECLVVGGRAMFRSDGYYRFMADADVVLESPVFTAGSQYNVVFETDALFIAPEISTVFEGDNIDTVVQTVANKVQAFNNRFVVYNAHVVDGAINVFCDYKFTCPAFLDGFPEWFSFCKNHFKDAGFVEFYDCIINSDGTFQTTFQPYLDLKPSVDAYLGPEIVRAVDGGRLWRTVINGISDAVNFCKNLRLHFDFGELKGTVVKRFKGILRTLLTVYNEFIQTTVSVLTICGISAKYYAFDKPMLCMHGITERVTSFDITSLGKPILDGLSRCNVFARGSTAVVVDSVDHELLDLEECDFVEPASHGVLVVSDDYGFYSCETGIYPYASDGKVLPLRFRKKAG

[0381] SEQ ID NO: 4: Amino acid sequence of TGEV NSP2

[0382] AIYVDQYMCGADGKPVIEGDFKDYFGDEDIIEFEGEEYHCAWTTVRDEKPLNQQTLFTIQEIQYNLDIPHKLPNCATRHVAPPVKKNSKIVLSEDYKKLYDIFGSPFMGNGDCLSKCFDTLHFIAATLRCPCGSESSGVGDWTGFKTACCGLSGKVKGVTLGDIKPGDAVVTSMSAGKGVKFFANCVLQYAGDVEGVSIWKVIKTFTVDETVCTPGFEGELNDFIKPESKSLVACSVKRAFITGDIDDAVHDCIITGKLDLSTNLFGNVGLLFKKTPWFVQKCGALFVDAWKVVEELCGSLTLTYKQIYEVVASLCTSAFTIVNYKPTFVVPDNRVKDLVDKCVKVLVKAFDVFTQIITIAGIEAKCFVLGAKYLLFNNALVKLVSVKILGKKQKGLECAFFATSLVGATVNVTPKRTETATISLNKVDDVVAPGEGYIVIVGDMAFYKSGEYYFMMSSPNFVLTNNVFKAVKVPSYDIVYDVDNDTKSKMIAKLGSSFEYDGDIDAAIVKVNELLIEFRQQSLCFRAFKDDKSIFVEAYFKKYKMPACLAKHIGLWNIIKKDSCKRGFLNLFNHLNELEDIKETNIQAIKNILCPDPLLDLDYGAIWYNCMPGCSDPSVLGSVQLLIGNGVKVVCDGCKGFANQLSKGYNKLCNAARNDIEIGGIPFSTFKTPTNTFIEMTDAIYSVIEQGKALSFRDADVPVVDNGTISTADWSEPILLEPAEYVKPKNNGNVIVIAGYTFYKDEDEHFYPYGFGKIVQRMYNKMGG

[0383] SEQ ID NO: 5: Amino acid sequence of PRCV NSP2

[0384] VIYVDQYMCGADGKPVIEGDFKDYFGDEDIIEFEGEEYHCAWTTVRDEKPLNQQTLFTIQEIQYNLDIPHKLPNCATRHVAPPVKKNSKIVLSEDYKKLYDIFGSPFMGNGDCLSKCFDTLHFIAATLRCPCGSESSGVGDWTGFKTACCGLSGKVKGVTLGDIKPGDAVVTSMSAGKGVKFFANCVLQYAGDVEGVSIWKVIKTFTVDETVCTLGFEGELNDFIKPESKSLIACSVKRAFITGEIDDAVHDCIITGKLDLSTNLFGNVGLLFKKPPWFVQKCGALFVDAWKVVEELCGSLTLTYKQIYEVVASLCNSVFTIVNYKPTFVVPDNRVKDLVDKCVKVLVKAFDVFTQNIIIAGIEAKCFVLGAKYLLFNNALVKLVSVKILGKKQKGLECAFFATSLVGATANVTPTRTETATISLNKVDDVVAPGEGYIVIVGDMAFYKSGEYYFMMSSPNSVLTNNVFKAVKVPSYDIVYDVDNDTKSKMIAKLGSSFEYDGDIDAAIVKVNELLIEFRQQSLCFRALKDDKTIFVEAYFKKYKMPACLANHIGLWNIIKKDSCKKGFLNLFNHLNELEDIKETNIQAIKNILCPDPLLDLDYGAIWYNCMPSCSDSSVLGSVQLLIGNGVKVVCDGCKGFANQLSKGYNKLCNAARNDIEIGGIPFSTFKTPTNTFIEMTDAIYSVVEQGEALSFRDADVPVVDDGTIATVDWSEPILLEPAEYVKPKNNGNVIVIAGYTFYKDEDEHFYPYGFGKIVQRMYNKMGG

[0385] SEQ ID NO: 6: Amino acid sequence of PHEV NSP2

[0386] VKPLLYVDQYGCDYTGGLADGLEAYADKTLQEMKALFPIWSQELPFDVTVAWHVVRDPRYVMRLQSASTIRSVAYVANPTEDLCDGSVVIKEPVHVYADDSIILRQHNLVDIMSCFYMEADAVVNAFYGVDLKDCGFVMQFGYIDCEQDLCDFKGWVPGNMIDGFACTTCGHVYETGDLLAQSSGVLPVNPVLHTKSAAGYGGFGCKDSFTLYGQTVVYFGGCVYWSPARNIWIPILKSSVKSYDSLVYTGVVGCKAIVKETNLICKALYLDYVEHKCGNLHQRELLGVSDVWHKQLLLNRGVYKPLLENIDYFNMRRAKFSLETFTVCADGFMPFLLDDLVPRAYYLAVSGHAFCDYADKLCHAVVSKSKDLLDVSLDSLGAAIHYLNFKIVDLAQHFSDFGASFVSTIVRYFKTFTTSTALAFAWVLFHVLHGAYIVVESDIYFVKNIPRYASAVAQAFRSVAKVVLDSLRVTFIDGLSCFKIGRRRICLSGRKIYEVERGLLHSSQLPLDVYDLTMPSQVQKAKQKPIYLKGSGSDFSLADSVVEVVTTSLTPCGYSEPPKFADKICIVDNVYMAKAGDKYYPVVVDDHVGLLDQAWRVPCA

[0387] SEQ ID NO: 7: Amino acid sequence of PDCoV NSP2

[0388] MAKNKSKRDAIALPENVPPPLQLFIHVAAAEEGHPKVTTYLGNYNLYATKAPPGVQVLSAKTSLTDFENVFGAQPTLRSIRNLVCEARSAEWTTSKNAFALKATQLDYSDAVLRAMIRFCPPKVSTLAAFALFGRLVKIEDKELAELARDTALELAYTAKIGISLADTRSVSLIHKDAYLTLSNEVVGVTFTAALMAKATTVNGAMQYSNFYLYPRATIKVTDGKAEAIATKPLPAATKGKPITEDVNLLPDYQQLLVDQVTGTEVKVGALTYVKTTDLPPLYFPKVKGGVVGIALKQQGTAAKKLNVVFHAQPDDVLLAFIQLQQFLNRTSDSSVEITDCQSYEVSPTVTVKIGPSKPGDVIVATDEEYLKCFETPEVGRLYKVFQTQSWAIIERSFSRIRVSKALSAFISFLQNLADNFTALSGVVTALIRELQDLTLDVATRITNIQFVYRAGKLIVDTTSVIAKLFQPFCDFISPFLRKVAGFAIYTVGNRMLMFTSTGTFLLTKATTKILNKAKYIFDVEPEYSVAVTTSKVVVHEALQQTDTKPTRALEAIDVVVGNTVLQMATDGTAFYPSDGTHASLPGFKAG

[0389] SEQ ID NO: 8: Complete coding sequence of SADS-CoV NSP2

[0390]

[0391] SEQ ID NO: 9: Complete coding sequence of TGEV NSP2

[0392]

[0393] SEQ ID NO: 10: Complete coding sequence of PRCV NSP2

[0394]

[0395] SEQ ID NO: 11: Complete coding sequence of PHEV NSP2

[0396]

[0397] SEQ ID NO: 12: Complete coding sequence of PDCoV NSP2

[0398]

Claims

1. A recombinant porcine coronavirus, the genome of which includes a complete deletion of the nonstructural protein 2 (NSP2) coding sequence.

2. The recombinant porcine coronavirus according to claim 1, wherein the deletion of the entire nonstructural protein 2 (NSP2) coding sequence is a complete deletion of the NSP2 gene.

3. The recombinant porcine coronavirus according to claim 1 or 2, wherein the virus is capable of infecting a host cell.

4. The recombinant porcine coronavirus according to any one of claims 1 to 3, wherein the recombinant porcine coronavirus is attenuated.

5. The recombinant porcine coronavirus according to any one of claims 1 to 4, wherein the porcine coronavirus is an alphacoronavirus, a betacoronavirus or a deltacoronavirus.

6. The recombinant porcine coronavirus of claim 5, wherein the porcine coronavirus is selected from porcine epidemic diarrhea virus (PEDV), porcine acute diarrhea syndrome-coronavirus (SADS-CoV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine delta coronavirus (PDCoV).

7. The recombinant porcine coronavirus of claim 6, wherein the porcine coronavirus is PEDV.

8. The recombinant porcine coronavirus of claim 7, wherein the porcine coronavirus is a genotype 2 PEDV.

9. The recombinant porcine coronavirus according to claim 8, wherein the recombinant porcine coronavirus is derived from the PEDV-ZJU / G2 / 2013 strain.

10. The recombinant porcine coronavirus of claim 6, wherein the porcine coronavirus is SADS-CoV.

11. The recombinant porcine coronavirus according to claim 8, wherein the recombinant porcine coronavirus is derived from the SADS-CoV-CH / GD-01 / 2017 strain.

12. The recombinant porcine coronavirus of any one of claims 1 to 11, wherein the NSP2 protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to any one of SEQ ID NOs: 1 and 3-7.

13. The recombinant porcine coronavirus of any one of claims 1 to 12, wherein the complete NSP2 coding sequence has a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to any one of SEQ ID NOs: 2 and 8-12.

14. A recombinant porcine coronavirus, in particular a recombinant porcine coronavirus according to any one of claims 1 to 9, which is i) the recombinant PEDV deposited with the CCTCC under accession number CCTCC V202263 on August 10, 2022, or any of its progeny or descendants; or ii) a recombinant PEDV, whose genomic sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity with the recombinant PEDV deposited with the CCTCC under the accession number CCTCC V202263 on August 10, 2022.

15. An isolated nucleic acid encoding the recombinant porcine coronavirus according to any one of claims 1 to 14.

16. A vector comprising the nucleic acid according to claim 15.

17. An immunogenic composition comprising the recombinant porcine coronavirus according to any one of claims 1 to 14, the isolated nucleic acid according to claim 15 or the vector according to claim 16.

18. The immunogenic composition of claim 17, further comprising a pharmaceutically acceptable carrier.

19. The immunogenic composition of claim 17 or 18, wherein the immunogenic composition is a vaccine.

20. The immunogenic composition of claim 19, wherein the vaccine further comprises an adjuvant.

21. The immunogenic composition according to any one of claims 17 to 20, for use i) Immunization of pigs; ii) eliciting an immune response against porcine coronavirus in pigs; iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or iv) Reducing mortality caused by porcine coronavirus infection in pigs.

22. The immunogenic composition for use according to claim 21, wherein the porcine coronavirus is PEDV, and / or the porcine coronavirus infection is a PEDV infection.

23. The immunogenic composition for use according to claim 21, wherein the porcine coronavirus is SADS-CoV, and / or the porcine coronavirus infection is SADS-CoV infection.

24. The immunogenic composition for use according to any one of claims 21 to 23, wherein the pig is a piglet or a sow.

25. The immunogenic composition according to any one of claims 17 to 20, for use i) reducing or preventing clinical symptoms or diseases caused by PEDV infection in piglets, wherein the piglets are to be suckled by sows to which the immunogenic composition according to any one of claims 15 to 18 has been administered; and / or ii) reducing mortality caused by PEDV infection in piglets, wherein the piglets are suckled by sows to which the immunogenic composition according to any one of claims 17 to 20 has been administered.

26. Use of the recombinant porcine coronavirus according to any one of claims 1 to 14, the isolated nucleic acid according to claim 15 or the vector according to claim 16 in the preparation of an immunogenic composition for i) Immunization of pigs; ii) eliciting an immune response against porcine coronavirus in pigs; iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or iv) Reducing mortality caused by porcine coronavirus infection in pigs.

27. The use according to claim 26, wherein the porcine coronavirus is PEDV, and / or the porcine coronavirus infection is PEDV infection.

28. The use according to claim 26, wherein the porcine coronavirus is SADS-CoV, and / or the porcine coronavirus infection is SADS-CoV infection.

29. The use according to any one of claims 26 to 28, wherein the pig is a piglet or a sow.

30. Use of the recombinant porcine coronavirus according to any one of claims 1 to 14, the isolated nucleic acid according to claim 15 or the vector according to claim 16 in the preparation of an immunogenic composition for i) reducing or preventing clinical symptoms or disease caused by PEDV infection in piglets, wherein the piglets are to be suckled by sows to which the immunogenic composition has been administered; and / or ii) reducing mortality caused by PEDV infection in piglets, wherein the piglets are suckled by sows to which the immunogenic composition has been administered.

31. The use according to any one of claims 26 to 30, wherein the immunogenic composition further comprises a pharmaceutically acceptable carrier.

32. The use according to any one of claims 26 to 31, wherein the immunogenic composition is a vaccine.

33. The use according to claim 32, wherein the vaccine further comprises an adjuvant.

34. A method for i) Immunization of pigs; ii) eliciting an immune response against porcine coronavirus in pigs; iii) reducing or preventing clinical signs or disease caused by porcine coronavirus infection in pigs; and / or iv) reducing mortality caused by porcine coronavirus infection in pigs, Wherein the method comprises administering to a pig an immunogenic composition according to any one of claims 17 to 20.

35. The method of claim 34, wherein the porcine coronavirus is PEDV and the porcine coronavirus infection is a PEDV infection.

36. The method of claim 34, wherein the porcine coronavirus is SADS-CoV, and / or the porcine coronavirus infection is SADS-CoV infection.

37. The method according to any one of claims 34 to 36, wherein the pig is a piglet or a sow.

38. A method for i) reducing or preventing clinical symptoms or disease caused by PEDV infection in piglets, and / or ii) reducing mortality caused by PEDV infection in piglets, wherein the piglets are suckled by sows to which the immunogenic composition according to any one of claims 17 to 20 has been administered.

39. A method for attenuating porcine coronavirus comprising deleting the entire nonstructural protein 2 (NSP2) coding sequence from its genome.

40. The method of claim 39, comprising deleting the entire NSP2 gene from its genome.

41. The method of claim 39 or 40, which produces an attenuated virus capable of infecting a host cell.

42. The method of any one of claims 39 to 41, wherein the porcine coronavirus is an alphacoronavirus, a betacoronavirus or a deltacoronavirus.

43. The method of claim 42, wherein the porcine coronavirus is selected from porcine epidemic diarrhea virus (PEDV), porcine acute diarrhea syndrome-coronavirus (SADS-CoV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine delta coronavirus (PDCoV).

44. The method of claim 43, wherein the porcine coronavirus is PEDV.

45. The method of claim 44, wherein the porcine coronavirus is genotype 2 PEDV.

46. ​​The method of claim 45, wherein the porcine coronavirus is PEDV strain PEDV-ZJU / G2 / 2013.

47. The method of claim 43, wherein the porcine coronavirus is SADS-CoV.

48. The method of claim 47, wherein the porcine coronavirus is SADS-CoV strain SADS-CoV-CH / GD-01 / 2017.

49. The method of any one of claims 39 to 48, wherein the NSP2 protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to any one of SEQ ID NOs: 1 and 3-7.

50. The method of any one of claims 39 to 49, wherein the complete NSP2 coding sequence has a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to any one of SEQ ID NOs: 2 and 8-12.

51. A method for producing a recombinant porcine coronavirus according to any one of claims 1 to 14, comprising introducing the isolated nucleic acid according to claim 15 or the vector according to claim 16 into a host cell, and The recombinant porcine coronavirus produced by the host cell is recovered.

52. The method of claim 51, wherein the host cell is a Vero cell, a ST cell or a PK cell.

53. A method for distinguishing an animal infected with porcine coronavirus from an animal vaccinated with an immunogenic composition according to any one of claims 17 to 20, comprising a) obtaining a sample, and b) testing the sample in an immunoassay and / or a genomic profiling assay.

54. The method of claim 53, comprising determining the presence of NSP2 coding sequences, NSP2 proteins, or antibodies against the NSP2 proteins in the sample.

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