Porcine epidemic diarrhea virus strain and immunogenic composition obtained therefrom

Abstract

To provide novel nucleotide and amino acid sequences of Porcine Epidemic Diarrhea Virus (PEDV), which are useful in the preparation of vaccines for treating and preventing diseases in swine and other animals, the vaccines being effective against multiple PEDV genotypes and isolates; to provide polynucleotide constructs that replicate in tissue culture and in host swine; and to provide novel full length PEDV genomes that can replicate efficiently in host animals and tissue culture.SOLUTION: Provided is an isolated Porcine Epidemic Diarrhea Virus encoded by a DNA polynucleotide having a particular sequence.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 62 / 121,955, filed Feb. 27, 2015; U.S. Provisional Patent Application No. 62 / 209,119, filed Aug. 24, 2015; U.S. Provisional Patent Application No. 62 / 250,961, filed Nov. 4, 2015; and U.S. Provisional Patent Application No. 62 / 276,022, filed Jan. 7, 2016, the entire contents of each of which are incorporated herein by reference.

[0002] The present invention relates to a novel immunogenic composition for protecting pigs from diseases caused by porcine epidemic diarrhea virus (PEDV).

Background Art

[0003] Porcine epidemic diarrhea (PED) is highly contagious and is characterized by dehydration, diarrhea, and high mortality in pigs, especially young piglets. The causative agent, porcine epidemic diarrhea virus (PEDV), is a single - stranded positive - sense RNA virus belonging to the genus Alphacoronavirus of the family Coronaviridae. PEDV has an overall genome size of approximately 28 kb and contains seven open reading frames. The symptoms of PEDV infection are often similar to those caused by transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV), which are also members of the family Coronaviridae. It should be noted that cross - protection between PEDV and TGEV is usually not observed, and at most, only about 60% similarity in the entire viral nucleotide sequence is observed.

[0004] PED was probably first observed in Europe around 1970 and later the causative virus was characterized (see, for example, M. Pensaert et al. Arch. Virol, v. 58, pp243 - 247, 1978, and D. Chasey et al., Res. Vet Sci, v. 25, pp255 - 256, 1978). PEDV had not been identified in North America until 2013, when a widespread pandemic began that year, causing severe economic losses to the swine industry. The virus appeared in multiple widely distributed groups of sows within days and spread to at least 32 states. Producers anticipate losses of up to 100% in naive newborn piglets. Current recommendations for infection control include implementation of strict biosecurity and / or intentional exposure of the entire herd to PEDV to achieve immunity.

[0005] PEDV caused widespread epidemics in several European countries during the 1970s - 1980s, but since the 1990s, PED has become less common, although it still occurs occasionally in Europe. This classical PEDV strain later spread to Asian countries such as Japan, China, and South Korea. Since 2010, severe epizootic PED pandemics have been reported in China, and the PEDV recovered from these pandemics was genetically different from the classical PEDV strain. The initial PED in US pigs had clinical findings similar to those observed in China. Sequence analysis revealed that the original US PEDV (hereinafter referred to as the US PEDV prototype strain) was most genetically similar to several PEDVs circulating in China during 2011 - 2012. In January 2014, a PEDV variant strain with insertions and deletions (INDELs) in the spike gene was identified in the US swine population compared to the US PEDV prototype strain. This variant strain was named the US PEDV S - INDEL variant. After the PED pandemic in the US, detections of the US prototype - type PEDV have been reported in China, Mexico, Taiwan, South Korea, and Japan, and the US S - INDEL variant PE DV detections have been reported in South Korea, Japan, Germany, Belgium, France, and Portugal. Currently, PEDV continues to be a significant threat to the global swine industry.

[0006] PEDV generally has poor growth in culture, and it is necessary to identify two specific strains suitable for culturing sufficient virus for the preparation of commercial vaccines. Furthermore, it is necessary to develop a vaccine that provides effective cross-protection against known PEDV isolates and is expected to provide effective cross-protection against PEDV strains evolving worldwide.

Summary of the Invention

[0007] The present invention encompasses an immunogenic composition comprising a mutant PEDV strain passaged from a prototype strain and an INDEL strain. The US PEDV S-INDEL mutant strain is genetically different from the US PEDV prototype strain and contains insertions and deletions characteristic of the S1 domain region of the spike protein, specifically the first 1170 bases thereof. The insertions and deletions include three deletions (a 1-nt deletion at position 167, an 11-nt deletion at position 176, and a 3-nt deletion at position 416), a 6-nt insertion between positions 474 and 475, and several other mutations mainly located in the first 1,170 nucleotides of the S1 region. The US PEDV S-INDEL mutant strain is less virulent than the US PEDV prototype strain and can be used as a live attenuated vaccine in one embodiment or for whole virus. Pigs infected with the continuously passaged live attenuated S-INDEL mutant strain of the present invention did not cause disease when administered to piglets and, importantly, showed cross-protection against challenge with either the virulent prototype PEDV strain or the S-INDEL mutant strain.

[0008] Accordingly, the present invention includes an immunogenic composition suitable for use as a vaccine comprising the S-INDEL-mutated PEDV strain of the present invention, or an immunogenic fragment thereof, which is preferably live and attenuated, one or more adjuvants, and optionally one or more excipients, in an amount effective to elicit the production of neutralizing antibodies in pigs. The adjuvant preferably provides an oil-in-water emulsion containing additional components. The immunogenic composition of the present invention protects pigs from infection by PEDV and is effective in a vaccination program including a single dose, a two-dose program, or multiple doses with a greater time interval, optionally from at least one week, optionally one month to several months. Note that depending on the level of risk of an outbreak in a particular pig population, a one-dose, two-dose, or multiple-dose vaccination program may sometimes be repeated as a preventive measure. Furthermore, vaccination of pregnant sows provides protection to piglets via maternal transfer of antibodies and T cells in colostrum and milk, although such protection may sometimes require additional vaccination of the piglets. Vaccination of all pigs, including piglets and adult pigs, is contemplated.

[0009] Surprisingly, it has been discovered that the US PEDV S-INDEL mutant strain of the present invention provides cross-reactivity / cross-protection against other PEDV strains, and protection is generally expected to be provided against recently emerging isolates in Europe, more specifically, isolates having a very high genetic similarity (>99%) to the US S-INDEL mutant strain at the whole-genome sequence level. Accordingly, the vaccine composition of the present invention is useful for protecting pigs from disease or for challenge by European strains of PEDV in general, including recent isolates.

[0010] The present invention includes novel nucleotide and amino acid sequences of PEDV (including its novel genotypes), all of which are useful for the preparation of vaccines for treating and preventing diseases in pigs and other animals. The vaccines provided by the implementation of the present invention are effective against multiple porcine PEDV genotypes and isolates. Similar to the infectious clones useful for virus propagation and vaccine preparation, diagnostic and therapeutic polyclonal and monoclonal antibodies are also features of the present invention. Particularly important is the use of the whole virus (live or attenuated), or most specifically the first 2.2 kb of the spike gene, more specifically a single antigen protein of the PEDV open reading frame from the S1 domain, and fragments of the full-length sequences encoding PEDV proteins as antigens. Vaccines are also disclosed that include these. The present invention also provides the full-length genomic sequences of different passage PEDV strains in cell culture that can replicate efficiently in host animals and tissue culture. Or most specifically, the first 2.2 kb of the spike gene, more specifically a single antigen protein of the PEDV open reading frame from the S1 domain, and fragments of the full-length sequences encoding PEDV proteins are also included in the disclosed vaccines.

[0011] The present invention provides a method for treating or preventing animal diseases or disorders caused by PEDV infection, including conditions directly caused by PEDV and conditions contributed to or promoted by PEDV. Porcine conditions that can be promoted by PEDV and can similarly be treated or prevented in accordance with the practice of the present invention include those caused by or related to transmissible gastroenteritis virus, PHEV, and PRCV.

[0012] The present invention also includes the option of administering a mixed vaccine, i.e., a combination of bivalent or multivalent antigens that may include live antigens, modified live antigens, or inactivated antigens against non-PEDV pathogens, together with appropriately selected adjuvants.

[0013] Based in part on the unique PEDV sequences disclosed herein, the present invention also provides a diagnostic kit for differentiating between pig animals inoculated with the above PEDV vaccine and pig animals infected with field strains of PEDV.

[0014] Representative embodiments of the present invention include isolated polynucleotide sequences that contain genomic polynucleotides encoding attenuated mutant PEDV proteins and can be used as immunogenic compositions. This can include all genomic sequences selected from the group consisting of: (a) SEQ ID NOs: 8 - 16, 35 - 39 encoding PEDV virus variants, or immunogenic fragments thereof, (b) Complementary strands of any of the sequences in (a), (c) Polynucleotides that hybridize to the sequences in (a) or (b) under stringent conditions defined as filter - bound DNA hybridization in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65°C and washing in 0.1×SSC / 0.1% SDS at 68°C, (d) Polynucleotides that are at least 70% identical to the polynucleotides in (a) or (b), (e) Polynucleotides that are at least 80% identical to the polynucleotides in (a) or (b), (f) Polynucleotides that are at least 90% identical to the polynucleotides in (a) or (b), and (g) Polynucleotides that are at least 95% identical to the polynucleotides in (a) or (b).

[0015] Preferably, it is combined with a second heterologous sequence. The present invention further provides vectors and plasmids for the expression of RNA and DNA molecules, their complements, fragments, and polynucleotides of any such RNA or DNA, and for PEDV viruses expressed from such nucleotide sequences, wherein the virus is live or fully or partially attenuated.

[0016] The present invention also provides vaccines that contain the aforementioned polynucleotide sequences and corresponding nucleotide sequences that can function as infectious clones. The present invention also provides mutant ta from PEDV as reflected in Table 1 attached hereto It also includes a nucleic acid ORF encoding a protein. The changes in Table 1 are described for the 5th passage of PEDV USA / IL20697 / 2014.

[0017]

Table 1-1

[0018]

Table 1-2

[0019] The present invention further provides nucleic acid sequences and the resulting protein variants that have amino acid substitutions, reduce virulence, result in attenuation, and enable the composition to be safely used as an immunogenic composition and as a vaccine.

[0020] The amino acid sequences and mutant proteins are also shown in Table 2 attached hereto, and the amino acid reference is made with respect to the 5th passage of PEDV / USA / IL20697 / 2014 is carried out.

[0021]

Table 2-1

[0022]

Table 2-2

[0023] In a further preferred embodiment, and using the substantial polypeptide sequence information disclosed herein, polypeptide vaccines are further provided wherein the antigen is defined by (a) the spike protein, or (b) an amino acid sequence that is at least 90 percent identical thereto, or (c) its arginine-rich region.

[0024] In a further embodiment, the present invention A live attenuated mutant porcine epidemic diarrhea virus (PEDV), and a vaccine composition comprising a carrier, said composition being capable of protecting pigs from challenge by both mutant and prototype strains of PEDV and of preventing or treating one or more of the symptoms associated with PEDV infection, the achievement of protection being determined by an endpoint selected from the group consisting of prevention or management of dehydration, fever, diarrhea, vomiting, reduced lactation ability, reduced reproductive ability, death, which are symptoms of PEDV infection, and prevention or management of weight loss or failure to gain weight, said strain encoding a protein having the following amino acid substitutions and their conservative variants, and a protein having a specific substitution and 99% homology to the rest of its sequence. The amino acid substitutions include the following or their conservative variants.

[0025] A) Passage P18R1 F6a Polyprotein 1a / 1b (SEQ ID NO: 46): P at position 551 (P immediately following DEDAT); Spike protein (SEQ ID NO: 47): P at position 1009 (P immediately following IGNIT), H at position 973 (H immediately following ALPFS), L at position 48 (L immediately following APAVV), V at position 345 (A immediately following TNLSF); ORF3 (SEQ ID NO: 48): deletion of I at position 144 (I immediately following YDGKS), deletion of 174 - 189 (immediately following LYLAI), LTANPL at positions 138 - 143 (immediately following NGKAA), and Nucleocapsid protein (SEQ ID NO: 51): H at position 27 (H immediately following LRVTN).

[0026] B) Passage P18R1 Gb8 Spike protein (SEQ ID NO: 47): P at position 1009 (P immediately following IGNIT), H at position 973 (H immediately following ALPFS), L at position 48 (L immediately following APAVV), V at position 345 (A immediately following TNLSF); ORF3 (SEQ ID NO: 48): deletion of I at position 144 (I immediately following YDGKS), deletion of 174 - 189 (immediately following LYLAI), LTANPL at positions 138 - 143 (immediately following NGKAA), and The H at position 27 of the nucleocapsid protein (SEQ ID NO: 51) (the H immediately after LRVTN).

[0027] C) Passage P7 Spike protein (SEQ ID NO: 47): K at position 633 (the K immediately after TPKPL); ORF3 (SEQ ID NO: 48): Deletion of L at position 189 (the L immediately after TANPL).

[0028] D) Passage P18 Spike protein (SEQ ID NO: 47): K at position 633 (the K immediately after TPKPL), R at position 884 (the R immediately after VYDPA); ORF3 (SEQ ID NO: 48): Deletion of L at position 189 (the L immediately after TANPL).

[0029] E) Passage P30 Spike protein (SEQ ID NO: 47): R at position 884 (the R immediately after VYDPA), A at position 959 (the A immediately after LIGGM); ORF3 (SEQ ID NO: 48): Deletion of L at position 189 (the L immediately after TANPL).

[0030] F) Passage P38 Spike protein (SEQ ID NO: 47): L at position 48 (the L immediately after APAVV), V at position 345 (after TNLSF), R at position 884 (the R immediately after VYDPA), A at position 959 (the A immediately after LIGGM), ORF3 (SEQ ID NO: 48): Deletion from positions 138 - 144 (after NGKAA), deletion of L at position 189 (the L immediately after TANPL), The H at position 27 of the nucleocapsid (SEQ ID NO: 51) (the H immediately after LRVTN).

[0031] G) Passage P45 Polyprotein 1a / 1b (SEQ ID NO: 46): M at position 1591 (the M immediately after VVKVS), S at position 5087 (the S immediately after YLFST), S at position 6138 (the S immediately after WQTFS); Spike protein (SEQ ID NO: 47): R at position 884 (R immediately after VYDPA), A at position 959 (A immediately after LIGGM), D at position 1225 (D immediately after IESLV); ORF3 (SEQ ID NO: 48): Deletion of Y at position 8 (Y immediately after LGLFO), deletion of positions 138 - 144 (immediately after NGKAA), and deletion of positions 174 - 189 (immediately after LYLAI), Envelope protein (SEQ ID NO: 49): F at position 62 (F immediately after YRVYK); Membrane protein I at position 183 (I immediately after IVYGG).

[0032] H) Passage P60 Polyprotein 1a / 1b (SEQ ID NO: 46): M at position 1591 (M immediately after VVKVS), S at position 5087 (S immediately after YLFST), S at position 6138 (S immediately after WQTFS); Spike protein (SEQ ID NO: 47): R at position 884 (after VYDPA), A at position 959 (after LIGGM), H at position 973 (H immediately after ALPFS), D at position 1225 (D immediately after IESLV); ORF3 (SEQ ID NO: 48): Deletion of Y at position 8 (Y immediately after LGLFO), deletion of positions 138 - 144 (immediately after NGKAA), and deletion of positions 174 - 189 (immediately after LYLAI), Envelope protein (SEQ ID NO: 49): F at position 62 (F immediately after YRVYK); Membrane protein A at position 5 (A immediately after MSNG), (I immediately after IVYGG at position 183).

[0033] I) Passage P3R1 Spike protein (SEQ ID NO: 47): L at position 48 (after APAVV), V at position 345 (V immediately after TNLSF); T at position 1272 (T immediately after DVFNA) Envelope protein (SEQ ID NO: 49): S at position 62 (S immediately after YRVYK); Nucleocapsid protein (SEQ ID NO: 51): H at position 27 (H immediately after LRVTN).

[0034] The present invention further includes passages and amino acid substitutions from a passage prototype virus deposited at the third passage of Genbank (registered trademark) accession number KF650371. The changes in amino acids are reflected as follows.

[0035] Polyprotein 1a / 1b (SEQ ID NO: 52): V at 814, A at 1076, F at 1564, I at 1896, H at 2310, Y at 2600, F at 3247, V at 3473, or R at 3522; Spike protein (SEQ ID NO: 54): N at 257, I at 326, F at 375, Y at 491, R at 881, R at 888, F at 1277, T at 1339, or L at 1358; Termination of ORF3 (SEQ ID NO: 55) at 39 or later; Position I at 69 of the envelope protein (SEQ ID NO: 56); Position T at 208 of the membrane protein (SEQ ID NO: 57); Positions L at 141, Q at 418, N at 424, I at 439 of the nucleocapsid protein (SEQ ID NO: 58).

[0036] Adding additional modifications to any PEDV passage described herein is within the scope of this specification, and said modifications include introducing mutations (prototypes, INDEL line 1, or INDEL line 2, and combinations thereof) of another passage described herein using known methods such as homologous recombination according to those skilled in the art.

[0037] GenBank (registered trademark) is a widely recognized U.S. NIH gene sequence database that includes a collection of publicly available annotated DNA sequences and further encompasses deposits from the European Molecular Biology Laboratory (EMBL) and the DNA DataBank of Japan (DDBJ): See Nucleic Acids Research, January 2013, v41(D1)D36-42 for consideration.

[0038] The following drawings form a part of this specification and are included to further illustrate certain embodiments or various aspects of the present invention. In some cases, embodiments of the present invention may be best understood by referring to the accompanying drawings in conjunction with the detailed description presented herein. The description and the accompanying drawings may emphasize certain specific examples or specific aspects of the present invention. However, those skilled in the art will understand that some of the examples or aspects may be used in combination with other examples or aspects of the present invention.

Brief Description of the Drawings

[0039]

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DETAILED DESCRIPTION OF THE INVENTION

[0040] The following definitions and introductory matters are applicable to this specification. The singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The word "or" means any one of the members of a particular list and also includes any combination of members of that list.

[0041] The term "adjuvant" refers to a compound that enhances the effect of a vaccine and can be added to a formulation containing an immunizing agent. An adjuvant provides enhancement of the immune response even after administration of a vaccine in a single dose only. Adjuvants can include, for example, muramyl dipeptide, pyridine, aluminum hydroxide, dimethyldioctadecylammonium bromide (DDA), oils, oil-in-water emulsions, saponins, cytokines, and other substances known in the art. Examples of suitable adjuvants are described in U.S. Patent Application No. US2004 / 0213817 A1. "Adjuvanted" refers to a compound incorporated with an adjuvant or a compound combined with an adjuvant.

[0042] "Antibody" refers to polyclonal and monoclonal antibodies, chimeric, as well as single-chain antibodies, and also includes Fab fragments, such as those produced by Fab or other immunoglobulin expression libraries. In relation to an antibody, the term "immunologically specific" refers to an antibody that binds to one or more epitopes of a target protein but does not substantially recognize or bind to other molecules in a sample containing a heterogeneous population of antigenic biomolecules.

[0043] As used herein, "attenuated" PEDV refers to a PEDV that is capable of infecting and / or replicating in a susceptible host, but is non-pathogenic or low-pathogenic to the susceptible host. For example, an attenuated virus may not cause observable / detectable clinical symptoms, or may cause fewer or less severe clinical symptoms, or may exhibit reduced viral replication efficiency and / or infectivity, compared to a related field isolate. Clinical symptoms of PEDV infection can include, but are not limited to, clinical diarrhea, vomiting, somnolence, deterioration of health, and dehydration.

[0044] An "epitope" is an immunologically active antigenic determinant in the sense that once administered to a host, it is capable of eliciting a humoral (B cell) and / or cellular (T cell) immune response. These are specific chemical groups or peptide sequences on a molecule that have antigenicity. Antibodies specifically bind to a particular antigenic epitope on a polypeptide. In animals, most antigens present several or even many antigenic determinants simultaneously. Such polypeptides can also be recognized as immunogenic polypeptides, and epitopes can be identified as further described.

[0045] As used herein, the term "immunogenic fragment" refers to a polypeptide or fragment that contains an allele-specific motif, epitope, or other sequence such that the polypeptide or fragment, when bound to an MHC molecule, induces a cytotoxic T lymphocyte ("CTL") response, and / or a B cell response (e.g., antibody production), and / or a helper T lymphocyte response, and / or a delayed-type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide or immunogenic fragment is derived. A DTH response is an immune reaction in which T cell-dependent macrophage activation and inflammation cause tissue damage. The DTH response to subcutaneous injection of an antigen is often used as an assay for cell-mediated immunity.

[0046] The term "induction of an immune defense response" means an (humoral and / or cellular) immune response that reduces or eliminates one or more of the symptoms of a disease, i.e., clinical signs, lesions, bacterial excretion, and bacterial replication, in the tissues of an infected subject as compared to a healthy control. Preferably, the reduction of said symptoms is statistically significant when compared to the control.

[0047] For the purposes of the present invention, an "infectious DNA molecule" is a DNA molecule that encodes the elements necessary for the replication, transcription, and translation of a virus into functional virus particles in a suitable host cell.

[0048] The term "isolated" is used to indicate that a cell, peptide, or nucleic acid has been separated from its natural environment. Isolated peptides and nucleic acids can be substantially pure, i.e., essentially free of other substances to which they may be bound in nature.

[0049] For the purposes of the present invention, the nucleotide sequence of a second polynucleotide molecule (either RNA or DNA) is "homologous" to the nucleotide sequence of a first polynucleotide molecule or has "identity" to said first polynucleotide molecule, and the nucleotide sequence of the second polynucleotide molecule encodes the same polyamino acid as the nucleotide sequence of the first polynucleotide molecule or encodes a polyamino acid that is sufficiently similar to the polyamino acid encoded by the nucleotide sequence of the first polynucleotide molecule so as to be useful in the practice of the present invention, based on the degeneracy of the genetic code. Homologous polynucleotide sequences also refer to both sense and antisense strands and, in all cases, to the complement of any such strand. For the purposes of the present invention, a polynucleotide molecule is useful in the practice of the present invention and is thus homologous or has identity, which can be used, for example, as a diagnostic probe for detecting the presence of PEDV or viral polynucleotides in a body fluid sample or tissue sample from an infected pig by standard hybridization techniques or amplification techniques. Usually, the nucleotide sequence of the second polynucleotide molecule is homologous to the nucleotide sequence of the first polynucleotide molecule when it has at least about 70% nucleotide sequence identity to the nucleotide sequence of the first polynucleotide molecule, based on the BLASTN algorithm (National Center for Biotechnology Information of the United States National Institute of Health (also known as NCBI) (Bethesda, Md., USA)). In a specific example of the calculations according to the practice of the present invention, BLASTP 2.2.6 [Tatusova TA and TL Madden, "BLAST 2 sequences--a When it has at least about 70% nucleotide sequence identity to the nucleotide sequence of the first polynucleotide molecule, it is homologous to the nucleotide sequence of the first polynucleotide molecule. In a specific example of the calculations according to the practice of the present invention, BLASTP 2.2.6 [Tatusova TA and TL Madden, "BLAST 2 sequences--a Reference is made to 「A new tool for comparing protein and nucleotide sequences.」(1999) FEMS Microbiol Lett. 174:247-250. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 0.1, and the 「blosum62」 scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad. Sci. USA 325 89:10915-10919. 1992). The percent identity is then calculated as follows: the total number of identical matches × 100 is divided by the length of the longer sequence + the number of gaps introduced into the longer sequence to align the two sequences.

[0050] Preferably, homologous nucleotide sequences have at least about 75% nucleotide sequence identity, and even more preferably, at least about 80%, 85%, 90%, and 95% nucleotide sequence identity. Because the genetic code is degenerate, homologous nucleotide sequences can contain any number of 「silent」 base changes, i.e., nucleotide substitutions that nevertheless encode the same amino acid.

[0051] The nucleotide sequence may further contain silent mutations, i.e., base substitutions, deletions, or additions that result in amino acid differences in the encoded polyamino acid, as long as the sequence maintains at least about 70% identity to the polyamino acid encoded by the first nucleotide sequence or is otherwise useful for practicing the invention. In this regard, certain conservative amino acid substitutions that are generally recognized not to inactivate the overall protein function may be made, for example, those relating to lysine, arginine, and histidine, which are positively charged amino acids (and vice versa), those relating to aspartic acid and glutamic acid, which are negatively charged amino acids (and vice versa), and certain groups of neutrally charged amino acids (in all cases, and vice versa), (1) alanine and serine, (2) asparagine, glutamine, and histidine, (3) cysteine and serine, (4) glycine and proline, (5) isoleucine, leucine, and valine, (6) methionine, leucine, and isoleucine, (7) phenylalanine, methionine, leucine, and tyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10) for example, those relating to tyrosine, tryptophan, and phenylalanine. Amino acids can be classified according to their physical properties and their contribution to protein secondary and tertiary structure. Thus, conservative substitutions are recognized in the art as the substitution of one amino acid for another having similar properties, and exemplary conservative substitutions are described in WO97 / 09433, page 10, published Mar. 13, 1997 (can be found in PCT / GB96 / 02197, filed Sep. 6, 1996). Conservative amino acids may be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY: NY (1975), pp. 71 - 77). Protein sequences can be aligned using multiple sequence alignments of both Vector NTI Advance 11.5 and CLUSTAL 2.1. As used herein, the recitation of a particular amino acid or nucleotide sequence shall include all silent mutations with respect to nucleic acid sequences and all conservatively modified variants with respect to amino acid sequences.

[0052] Identical nucleotide sequences can be determined by comparison of nucleotide sequences, for example, by using BLASTN as described above. Alternatively, identical nucleotide sequences can be determined by hybridization under selected conditions. For example, the nucleotide sequence of a second polynucleotide molecule is under moderately stringent conditions, for example, 0.5 M NaHPO at 65 °C 4, hybridization with filter-bound DNA in 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and washing in 0.2× SSC / 0.1% SDS at 42° C. (see Ausubel et al editors, Protocols in Molecular Biology, Wiley and Sons, 1994, pp. 6.0.3 to 6.4.10), or under conditions that otherwise result in hybridization to the complement of SEQ ID NO:1 of a sequence encoding a PEDV virus as defined hereinafter, is homologous to SEQ ID NO:1 (or any other specific polynucleotide sequence) when hybridizing to the complement of SEQ ID NO:1. Alterations in hybridization conditions can be determined empirically or can be calculated precisely based on the length and percentage of guanosine / cytosine (GC) base pairs in the probe. Hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.

[0053] In another embodiment, the second nucleotide sequence hybridizes to the complement of SEQ ID NO:1 under highly stringent conditions, for example, in 0.5 M NaHPO at 65° C., as known in the art 4 , hybridization with filter-bound DNA in 7% SDS, 1 mM EDTA, and washing in 0.1× SSC / 0.1% SDS at 68° C., is homologous to SEQ ID NO:1 (or any other sequence of the invention) when hybridizing to the complement of SEQ ID NO:1.

[0054] It should be further understood that the isolated polynucleotide molecules and isolated RNA molecules of the present invention include both synthetic molecules and molecules obtained by recombinant techniques such as cloning and transcription in vitro.

[0055] Many of the vaccine - capable attenuated PEDV viruses of the present invention result in a substantial deletion of the ORF3 protein that occurs as a result of an attenuating mutation in the ORF3 nucleotide sequence, which causes substantial internal deletions, and / or, most typically, the occurrence of short translations due to frameshifts and the appearance of stop codons. It should be noted that within the practice of the present invention, the calculation of alignments and percent identities, and the resulting identities described (whether nucleotide or amino acid sequence), can be calculated with or without reference to the deleted ORF3 sequence.

[0056] "Mammal" includes any warm - blooded vertebrate of the class Mammalia, including humans. "Pharmaceutically acceptable carrier" means any conventional pharmaceutically acceptable carrier, vehicle, or excipient used in the art for the manufacture and administration of vaccines. A pharmaceutically acceptable carrier is typically a non - toxic, inert, solid, or liquid carrier.

[0057] The terms "porcine" and "swine" are used interchangeably herein and refer to any animal that is a member of the family Suidae, such as a pig.

[0058] As used herein, a "susceptible" host refers to a cell or animal that can be infected by PEDV. When introduced into a susceptible animal, the attenuated PEDV may also induce an immunological response against PEDV or its antigen, thereby conferring immunity on the animal against PEDV infection. to PEDV infection.

[0059] The term "vaccine" refers to an antigen preparation that produces immunity against a disease in order to prevent or ameliorate the effects of infection. Vaccines are typically prepared by combining an immunologically effective amount of an immunogen with an adjuvant effective to enhance the immune response of the subject being vaccinated to the immunogen.

[0060] The vaccine formulation contains an "therapeutically effective amount" of an active ingredient, i.e., an amount capable of inducing an immune defense response in the subject to which the composition is administered. In the treatment and prevention of PEDV disease, for example, an "therapeutically effective amount" is preferably an amount that enhances the resistance of the vaccinated subject to new infections and / or reduces the clinical severity of the disease. Such protection is indicated by either a reduction or absence of the symptoms normally exhibited by a subject infected with PEDV, and / or a decrease in the viral particle count. The vaccine can be administered prior to infection as a preventive measure against PEDV. Alternatively, the vaccine may be administered after the subject has already contracted the disease. A vaccine administered after exposure to PEDV may be able to reduce the disease and elicit an immune response superior to that of the natural infection itself.

[0061] For the implementation of all aspects of the present invention, it is well known to those skilled in the art that there is no absolute immunological boundary in immunological assays with respect to animals that are seronegative to exposure to a particular antigen or pathogen and animals that are seropositive (having been exposed to the vaccine or pathogen). Nevertheless, those skilled in the art will recognize that in a serum neutralization assay, seropositive animals are typically detected at a binding dilution of at least 1:1000 at most, while seronegative animals are predicted not to neutralize at a higher dilution of about 1:20 or 1:10. Vaccine formulation / Immunogenic composition The present invention also relates to an immunogenic composition suitable for use as a vaccine, comprising a mutant PEDV strain according to the present invention. The immunogenic composition according to the present invention elicits a specific humoral immune response against PEDV, including neutralizing antibodies.

[0062] A preferred immunogenic composition based on the mutant strains disclosed herein can provide a live attenuated virus that exhibits high immunogenicity while not causing dangerous pathogenic or lethal effects.

[0063] However, the immunogenic composition of the present invention is not limited to any particular type or preparation method. These include, but are not limited to, infectious DNA vaccines (i.e., using plasmids, vectors, or other conventional carriers for direct injection of DNA into pigs), live vaccines, modified live vaccines, inactivated vaccines, subunit vaccines, attenuated vaccines, recombinant gene vaccines, and the like. These vaccines are prepared by standard methods known in the art.

[0064] The present invention preferably includes a vaccine composition comprising the attenuated live mutant PEDV of the present invention and a pharmaceutically acceptable carrier. As used herein, the expression "attenuated live PEDV of the present invention" encompasses any attenuated live PEDV strain comprising one or more of the changes described herein. A pharmaceutically acceptable carrier can be, for example, water, a stabilizer, a preservative, a culture medium, or a buffer, etc. The vaccine preparation containing the attenuated PEDV of the present invention can be prepared in the form of a liquid suspension or a freeze-dried form, or alternatively in a frozen form. When frozen, glycerol or other similar substances may be added to enhance stability upon freezing. The advantages of attenuated live vaccines generally include presenting the immunogenic determinants of all relevant infectious agents in their natural form to the host immune system, and requiring a relatively small amount of immunizing agent due to the ability of the agent to multiply in the vaccinated host.

[0065] Attenuation of the live vaccine virus to a level of pathogenicity insufficient to cause adverse effects in the vaccinated target animal can preferably be achieved by known procedures including serial passage. The following references provide various general methods for the attenuation of coronaviruses and are suitable for the attenuation or further attenuation of any of the strains useful in the practice of the present invention: B. Neuman et al., Journal of Virology, vol. 79, No. 15, pp. 9665-9676, 2005; J. Netland et al., Virology, v 399(1), pp. 120-128, 2010; Y-P Huang et al., "Sequence changes of infectious bronchitis virus isolates in the 3’ 7.3kb of the genome after attenuating passage in embryonated eggs, Avian Pathology, v. 36(1), (Abstract), 2007; and S. Hingley et al., Virology, v. 200(1) 1994, pp. 1-10; (see U.S. Patent No. 3,914,408); and Ortego et al., Virology, vol. 308(1), pp. 13-22, 2003.

[0066] Further recombinant vaccines desirable in the present invention are produced by techniques known in the art. Such techniques include, but are not limited to, further manipulation of recombinant DNA, modification of the amino acid sequence of a recombinant protein, or substitution into the amino acid sequence.

[0067] A recombinant DNA technology-based recombinant vaccine is produced by identifying an alternative portion of a viral gene encoding a protein (e.g., a protein derived from M, GP2, GP3, GP4, or GP5, etc.) involved in inducing a more potent immune response or defensive response in, for example, pigs. Various subtypes and isolates of viral protein genes can be subjected to the DNA shuffling method. The resulting heterologous chimeric viral protein can be used for a broadly protective subunit vaccine. Alternatively, such a chimeric viral gene or immunodominant fragment can be cloned into a standard protein expression vector such as a baculovirus vector and used to infect appropriate host cells (see, for example, O’Reilly et al., “Baculovirus Expression Vectors: A Lab Manual,” Freeman & Co., 1992). The host cells are cultured and thus express the desired vaccine protein, which can be purified to the desired extent and formulated into a suitable vaccine product.

[0068] If the clone retains any undesirable natural ability to cause disease, it is also possible to identify the nucleotide sequence of the viral genome involved in any residual virulence and, for example, genetically engineer the virus to be non-toxic by site-directed mutagenesis. Site-directed mutagenesis can add, delete, or alter one or more nucleotides (see, for example, Zoller et al., DNA 3:479-488, 1984). An oligonucleotide containing the desired mutation is synthesized and annealed to a portion of the single-stranded viral DNA. The hybrid molecule resulting from that procedure is used to transform bacteria. Then, the isolated double-stranded DNA containing the appropriate mutation is ligated to the latter restriction enzyme fragment that is later transfected in a suitable cell culture to generate the full-length DNA. Ligation of the genome into a suitable vector for transfer can be accomplished by any standard technique known to those skilled in the art. Transfection of the vector into a host cell to produce progeny virus can be done using any of the conventional methods such as calcium phosphate or DEAE-dextran mediated transfection, electroporation, protoplast fusion, and other well-known techniques (see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press, 1989). The cloned virus then exhibits the desired mutation. Alternatively, two oligonucleotides containing the appropriate mutation may be synthesized. These can be annealed to form double-stranded DNA, which can be inserted into the viral DNA to produce the full-length DNA.

[0069] An immunologically effective amount of the vaccine of the present invention is administered to pigs in need of protection from viral infection. The immunologically effective amount or immunogenic amount for inoculating pigs can be readily determined by routine testing or can be easily titrated. The effective amount is the amount at which a sufficient immunological response to the vaccine is obtained to protect pigs exposed to the PEDV virus. Preferably, the pigs are protected to such an extent that any deleterious physiological symptoms or effects of the viral disease are significantly reduced, improved, or completely prevented.

[0070] The vaccine of the present invention can be formulated according to accepted practices that are acceptable because it contains carriers acceptable to animals, such as standard buffers, stabilizers, diluents, preservatives, and / or solubilizing agents, and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include, inter alia, sodium chloride, dextrose, mannitol, sorbitol, and lactose. Stabilizers include, inter alia, albumin. Other suitable vaccine vehicles and additives, including those particularly useful in the formulation of modified live vaccines, are known to those skilled in the art or will become apparent. See, for example, Remington’s Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.

[0071] The vaccine of the present invention may further contain one or more additional immunomodulatory components, such as, for example, an adjuvant or a cytokine. Non-limiting examples of adjuvants that can be used in the vaccine of the present invention include RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide, oil-in-water emulsions, water-in-oil emulsions, for example, Freund's complete and incomplete adjuvants, block copolymers (CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.), Amphigen® adjuvant, saponin, Quil A, or other saponin fractions, monophosphoryl lipid A, ionic polysaccharides, and abridine lipid-amine adjuvants. Non-limiting examples of oil-in-water emulsions useful in the vaccine of the present invention include modified SEAM62 and SEAM1 / 2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v / v) squalene (Sigma), 1% (v / v) SPAN® 85 detergent (ICI surfactant), 0.7% (v / v) TWEEN® 80 detergent (ICI surfactant), 2.5% (v / v) ethanol, 200 μg / ml Quil A, 100 μg / ml cholesterol, and 0.5% (v / v) lecithin. Modified SEAM1 / 2 is an oil-in-water emulsion containing 5% (v / v) squalene, 1% (v / v) SPAN® 85 detergent, 0.7% (v / v) Tween 80 detergent, 2.5% (v / v) ethanol, 100 μg / ml Quil A, and 50 μg / ml cholesterol. Other immunomodulators that can be included in the vaccine include, for example, one or more interleukins, interferons, or other known cytokines.

[0072] With additional adjuvant systems, combinations of epitopes of both helper T cells and B cells It becomes possible to conjugate, resulting in one or more types of covalently linked T-B epitope linkage structures, which can be further lipidated, such as those described in WO2006 / 084319, WO2004 / 014957, and WO2004 / 014956.

[0073] In a preferred embodiment of the present invention, the ORF1 PEDV protein, or other PEDV proteins, or fragments thereof are formulated with 5% AMPHIGEN® as described hereinafter. Adjuvant composition The vaccine composition of the present invention may or may not contain an adjuvant. Specifically, in the case of an orally infective virus, the modified live vaccine of the present invention may be used without an adjuvant using a sterile carrier. Adjuvants that can be used for oral administration include those based on CT-like immunomodulatory components (rmLT, CT-B, i.e., recombinant mutant heat-labile toxin of E. coli, cholera toxin B subunit), or those using polymers and alginic acid, or mucosal adhesives such as chitosan, or encapsulation by liposomes. The preferred dosage of an adjuvanted or non-adjuvanted vaccine at the minimum protective dose by vaccine release is about 10 to about 10 6 log 10 TCID 50 or more viruses can be provided. "TCID50" refers to the "tissue culture infectious dose" and is defined as the dilution rate of the virus required to infect 50% of the inoculated cell culture of a given batch. Various methods, including the Spearman-Karber method used throughout this specification, can be used to calculate the TCID50. For an explanation of the Spearman-Karber method, see B.W. Mahy & H.O. Kangro, Virology Methods Manual, p. 25-46 (1996). The adjuvant, if present, may be provided as an emulsion and, more generally, should not decrease by more than 0.7 log (80% decrease) of the starting titer when parenteral administration is selected.

[0074] In one example, the adjuvant component is provided from a combination of lecithin in light mineral oil and an aluminum hydroxide component. Details regarding the composition and formulation of Amphigen® (as a representative lecithin / mineral oil component) are as follows.

[0075] Preferred adjuvantation can be provided as a 2 ML dose in a buffer further containing about 5% (v / v) Rehydragel® (aluminum hydroxide gel) and "20% Amphigen"® at a final concentration of about 25% (v / v). Amphigen® is generally described in U.S. Patent No. 5,084,269 and provides deoiled lecithin (preferably soy) dissolved in light oil and then dispersed as an oil-in-water emulsion in an aqueous solution or suspension of an antigen. Amphigen has been improved to provide a so-called "20% Amphigen" component for use in the final adjuvanted vaccine composition of the present invention according to the protocol of U.S. Patent No. 6,814,971 (see columns 8 - 9 thereof). Thus, a stock mixture of 10% lecithin and 90% carrier oil (DRAKEOL®, Penreco, Karns City, PA) is diluted 1:4 with 0.63% phosphate buffered saline, thereby reducing the lecithin and DRAKEOL components to 2% and 18% respectively (i.e., 20% of their original concentrations). Tween 80 and Span 80 surfactants are added to the composition, with representative and preferred final amounts of 5.6% (v / v) Tween 80 and 2.4% (v / v) Span 80, where Span is initially provided in the stock DRAKEOL component and Tween is initially provided from the buffered saline component, and the mixture of saline and DRAKEOL component ultimately results in the desired surfactant concentration. The mixture of DRAKEOL / lecithin and saline can be obtained using a Model 405 In-Line Slim Emulsifier device (Charles Ross and Son, Hauppauge, NY, USA).

[0076] The vaccine composition also contains Rehydragel® LV (about 2% aluminum hydroxide content in the stock material) as an additional adjuvant component (available from Reheis, NJ, USA and ChemTrade Logistics, USA). Further dilution with 0.63% PBS results in the final vaccine composition containing the following amounts of components per 2ML dose: 5% (v / v) Rehydragel® LV, 25% (v / v) of "20% Amphigen" (i.e., further diluted 4-fold), and 0.01% (w / v) merthiolate.

[0077] As understood in the art, the order of addition of components may vary to provide an equivalent final vaccine composition. For example, a suitable dilution of the virus can be prepared in the buffer. Then, an appropriate amount of Rehydragel® LV stock solution (about 2% aluminum hydroxide content) may be added with mixing to achieve the desired 5% (v / v) concentration of Rehydragel® LV in the actual final product. Once prepared, this intermediate stock material can be combined with an appropriate amount of "20% Amphigen" stock (already containing the required amounts of Tween 80 and Span 80 as outlined above) to obtain a final product with 25% (v / v) of "20% Amphigen" again. An appropriate amount of 10% merthiolate may finally be added.

[0078] The vaccine composition of the present invention allows for variation in all components such that the total dose of antigen can preferably vary by up to 100-fold (higher or lower) compared to the aforementioned antigen dose, and most preferably by up to 10-fold (higher or lower). Similarly, the surfactant concentration (either Tween or Span) may vary up to 10-fold independently of each other, or they may be replaced by similar materials at appropriate concentrations well understood in the art or completely removed.

[0079] The concentration of Rehydragel® in the final product may be varied by initially using equivalent materials available from many other manufacturers (i.e., Alhydrogel®, Brenntag; Denmark), or by further variations of the Rehydragel® product line, such as the use of CG, HPA, or HS. Using LV as an example, its final effective concentration, including 0% to 20%, is more preferably 2% to 12%, and most preferably 4% to 8%. Similarly, the final concentration of Amphigen (expressed as % of “20% Amphigen”) is preferably 25%, although this amount may vary from 5% to 50%, preferably 20% to 30%, and most preferably about 24% to 26%.

[0080] According to the practice of the present invention, the oil used in the adjuvant formulation of the present invention is preferably mineral oil. As used herein, the term “mineral oil” refers to a mixture of liquid hydrocarbons obtained from petroleum by distillation techniques. This term is synonymous with “liquid paraffin,” “liquid petrolatum,” and “white mineral oil.” This term is also intended to include “light mineral oil,” i.e., an oil that is similarly obtained by distillation of petroleum but has a specific gravity slightly lower than that of white mineral oil. See, for example, Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, at pages 788 and 1323). Mineral oil can be obtained from various commercial sources, such as J.T. Baker (Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). A preferred mineral oil is a light mineral oil marketed under the name DRAKEOL®.

[0081] Typically, the oily phase is present in an amount of 50% to 95% by volume, preferably in an amount of more than 50% to 85%, more preferably in an amount of more than 50% to 60%, and even more preferably in an amount of more than 50 to 52% v / v of the vaccine composition. The oily phase contains an oil and an emulsifier (e.g., SPAN® 80, TWEEN® 80, etc.) (if any such emulsifier is present).

[0082] Suitable non-natural synthetic emulsifiers for use in the adjuvant formulations of the present invention are sorbitan-based nonionic surfactants, such as fatty acid-substituted sorbitan surfactants (commercially available under the name of SPAN® or ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®); polyethoxylated fatty acids (e.g., stearic acid available under the name of SIMULSOL® M-53), polyethoxylated isooctylphenol / formaldehyde polymers (TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®); polyoxyethylene nonylphenyl ether (TRITON® N), polyoxyethylene isooctylphenyl ether (TRITON® X) are also included. Preferred synthetic surfactants are surfactants available under the names of SPAN® and TWEEN®, such as TWEEN®-80 (polyoxyethylene (20) sorbitan monooleate) and SPAN®-80 (sorbitan monooleate). Generally, the emulsifier(s) can be present in the vaccine composition in an amount of 0.01% to 40% by volume, preferably 0.1% to 15%, more preferably 2% to 10%.

[0083] In an alternative embodiment of the present invention, the final vaccine composition contains SP-Oil® and Rehydragel® LV as adjuvants (or other Rehydragel® or Alhydrogel® products), and the preferred amounts are about 5-20% SP-Oil (v / v) and about 5-15% Rehydragel LV (v / v), with 5% and 12% being the most preferred amounts, respectively. In this regard, it should be understood that the % of Rehydragel refers to the dilution rate from the commercially available stock product. (SP-Oil® is a flowing oil emulsion containing polyoxyethylene-polyoxypropylene block copolymer (Pluronic® L121, BASF Corporation), squalene, polyoxyethylene sorbitan monooleate (Tween® 80, ICI Americas) and a buffered salt solution.) It should be noted that the present invention can also be successfully carried out using the condition that the adjuvant component is only Amphigen®.

[0084] In another embodiment of the present invention, the final vaccine composition contains TXO as an adjuvant; TXO is outlined in WO2015 / 042369. All TXO compositions disclosed in that patent are useful for the preparation of the vaccines of the present invention. In TXO, the immunostimulatory oligonucleotide ("T"), preferably the ODN, preferably contains a palindromic sequence with an optionally modified backbone and is present in an amount of 0.1-5 μg per 50 μl of the vaccine composition (e.g., 0.5-3 μg per 50 μl of the composition, more preferably 0.09-0.11 μg per 50 μl of the composition). Its preferred species is SEQ ID NO: 8 as listed in the WO2015 / 042369 publication (PCT / US2014 / 056512) (page 17). The polycationic carrier ("X") is present in an amount of 1-20 μg per 50 μl (e.g., 3-10 μg per 50 μl, or about 5 μg per 50 μl). Light mineral oil ("O") is also a component of the TXO adjuvant.

[0085] In certain embodiments, the TXO adjuvant is prepared as follows: a) Dissolve sorbitan monooleate, MPL-A, and cholesterol in light mineral oil. Sterile filter the resulting oil preparation. b) Dissolve the immunostimulatory oligonucleotide, dextran DEAE, and polyoxyethylene (20) sorbitan monooleate in the aqueous phase to form an aqueous solution. c) Add the aqueous solution to the oil preparation while continuously homogenizing to form the adjuvant formulation TXO.

[0086] All adjuvant compositions of the invention can be used with any of the PEDV strains and isolates encompassed herein. Additional adjuvants useful in the practice of the invention include Prezent-A (see generally U.S. Patent Application Publication No. US20070298053) and "QCDCRT" or "QCDC" type adjuvants (see generally U.S. Patent Application Publication No. US20090324641). Excipient The immunogenic vaccine composition of the present invention can further contain a pharmaceutically acceptable carrier, excipient, and / or stabilizer in the form of a lyophilized formulation or an aqueous solution (see, for example, Remington: The Science and practice of Pharmacy, 2005, Lippincott Williams). The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at its dosage and concentration, and includes buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives ((((o-carboxyphenyl)thio)ethyl mercury sodium salt (THIOMERSAL), octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; saccharides such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG), TWEEN, or PLURONICS.

[0087] The vaccine of the present invention can optionally be formulated for slow release of the virus, infectious DNA molecule, plasmid, or viral vector of the present invention. Examples of such slow release formulations include complexes of biocompatible polymers, such as those combined with poly(lactic acid), lactic acid (glycolic acid copolymer), methylcellulose, hyaluronic acid, collagen, etc., and include a virus, infectious DNA molecule, plasmid, or viral vector. The structure, selection, and use of degradable polymers in drug delivery vehicles are outlined in several publications, including A. Domb et al., 1992, Polymers for Advanced Technologies 3:279-292, which is incorporated herein by reference. Further guidance on the selection and use of polymers in pharmaceutical formulations can be found in textbooks known in the art, such as M. Chasin and R. Langer (eds), 1990, "Biodegradable Polymers as Drug Delivery Systems" in: Drugs and the Pharmaceutical Sciences, Vol. 45, M. Dekker, NY. Alternatively or additionally, the virus, plasmid, or viral vector can be microencapsulated to improve administration and efficacy. Methods for microencapsulating antigens are well known in the art and include, for example, the techniques described in U.S. Patent No. 3,137,631, U.S. Patent No. 3,959,457, U.S. Patent No. 4,205,060, U.S. Patent No. 4,606,940, U.S. Patent No. 4,744,933, U.S. Patent No. 5,132,117, and International Patent Publication No. WO95 / 28227 (all of which are incorporated herein by reference).

[0088] Liposomes can also be used to provide sustained release of viruses, plasmids, viral proteins, or viral vectors. Details regarding methods of making and using liposome formulations can be found, inter alia, in U.S. Patent Nos. 4,016,100, 4,452,747, 4,921,706, 4,927,637, 4,944,948, 5,008,050, and 5,009,956, all of which are incorporated herein by reference.

[0089] An effective amount of any of the above vaccines can be determined by conventional means starting with a low dose of virus, viral protein, plasmid, or viral vector and then increasing the dosage while monitoring the effect. An effective amount can be obtained after a single administration of the vaccine or after multiple administrations of the vaccine. When determining the optimal dosage per animal, known factors can be taken into account. These include the species, size, age, and general condition of the animal, the presence of other drugs in the animal, etc. The actual dosage is preferably selected after considering the results of other animal experiments.

[0090] One way to detect whether an appropriate immune response has been achieved is to determine seroconversion and antibody titers in the animal after vaccination. The timing of vaccination and, if any, the number of booster administrations are preferably determined by a physician or veterinarian based on an analysis of all relevant factors, some of which are described above.

[0091] An effective dosage of the virus, protein, infectious nucleotide molecule, plasmid, or viral vector of the present invention can be determined using known techniques taking into account factors that can be determined by one of ordinary skill in the art such as the body weight of the animal to be vaccinated. The dosage of the virus of the present invention in the vaccine of the present invention is preferably about 10 1 ~ about 10 9 pfu (plaque forming units), more preferably about 10 2 ~ about 10 8pfu, most preferably about 10 3 ~ about 107 pfu. The dosage of the plasmid of the present invention in the vaccine of the present invention is preferably in the range of about 0.1 μg to about 100 mg, more preferably about 1 μg to about 10 mg, and even more preferably about 10 μg to about 1 mg. The dosage of the infectious DNA molecule of the present invention in the vaccine of the present invention is preferably in the range of about 0.1 μg to about 100 mg, more preferably about 1 μg to about 10 mg, and even more preferably about 10 μg to about 1 mg. The dosage of the viral vector of the present invention in the vaccine of the present invention is preferably about 10 1 pfu ~ about 10 9 pfu, more preferably about 10 2 pfu ~ about 10 8 pfu, even more preferably about 10 3 ~ about 10 7 pfu. Suitable dosage sizes are in the range of about 0.5 ml to about 10 ml, more preferably about 1 ml to about 5 ml.

[0092] Suitable dosages for the viral protein or peptide vaccine according to the practice of the present invention are usually in the range of 1 to 50 micrograms per dose or more than that which can be determined by standard methods, and the amount of adjuvant is determined by the methods recognized for each such substance. In a preferred example of the present invention related to swine vaccination, the optimal age target for the animal is about 1 day to 21, which can also accommodate other scheduled vaccinations such as vaccines against Mycoplasma hyopneumoniae before weaning. Further, the preferred schedule for a vaccine for breeding sows includes similar dosages using an annual revaccination schedule. Dosage Preferred clinical applications are treatment, management, and prevention in both breeding sows and gilts prior to farrowing, and subsequent vaccination of piglets. In a representative example (applicable to both sows and gilts), two 2 mL doses of vaccine are used, although of course the actual volume of the dose is a function of how the vaccine is formulated using an actual dose in the range of 0.1 - 5 mL, taking into account the size of the animal. Single-dose vaccination is also appropriate.

[0093] The first dose may be administered as early as before breeding up to 5 weeks prior to farrowing, and the second dose is preferably administered about 1 - 3 weeks prior to farrowing. The dose of the vaccine preferably provides an amount of virus material corresponding to about 10 6 ~10 8 and more preferably about 10 7 ~10 7.5 TCID 50 (tissue culture infectious dose) and may be further varied as recognized in the art. Booster doses may be administered 2 - 4 weeks prior to any subsequent farrowing. Intramuscular vaccination (all doses) is preferred, although one or more of the doses may be administered subcutaneously. Oral administration is also preferred. Vaccination may also be effective in naïve and non-naïve animals as achieved by either planned or natural infection.

[0094] In a further preferred example, sows or gilts are vaccinated intramuscularly or orally 5 weeks before parturition and then 2 weeks before parturition. Under these conditions, antibodies with neutralizing activity are generated in vaccinated sows negative for PEDV (neutralization titer measured by fluorescent focus from serum samples), and since these antibodies are passively transferred to piglets, a protective immune response can be demonstrated. The protocol of the present invention is also applicable to the treatment of sows and gilts that are already seropositive, as well as piglets and breeding pigs. Also, booster vaccinations may be performed, and they may be via different administration routes. It is preferred to re-vaccinate the sow again before any subsequent parturition, but even if the sow has only been vaccinated in relation to the previous parturition, the vaccine composition of the present invention can still provide protection to the piglets via continuous passive transfer of antibodies.

[0095] Thereafter, it should be noted that piglets may be vaccinated as early as on the first day of birth. For example, especially when the dam was vaccinated before insemination but not before farrowing, the piglets are vaccinated on the first day, and a booster dose may or may not be given at 3 weeks of age. Vaccination of piglets can also be effective when the dam was not previously naïve due to either natural or planned infection. Vaccination of piglets can be effective even when the dam has not been previously exposed to the virus or was not vaccinated before farrowing. Breeder pigs (typically raised for breeding purposes) should be vaccinated once every six months. Variations in dosage are within the practice of the art. It should be noted that the vaccine of the present invention is safe for use in pregnant animals (throughout all gestational periods) and neonatal pigs. The vaccine of the present invention is attenuated to an acceptable safety level (i.e., no deaths are seen and only mild transient clinical signs, singular or plural, normal for neonatal pigs) even in the most susceptible animals (including neonatal pigs in this case). Of course, from the perspective of protecting pig herds from both PEDV epidemics and the occurrence of low-level persistent PEDV, a continuous vaccination program for multiparous sows is very important. It should be recognized that sows or gilts immunized with PEDV MLV passively transfer immunity, including PEDV-specific IgA, to piglets and protect the piglets from PEDV-related diseases and death. Furthermore, usually, pigs immunized with PEDV MLV excrete less amount and / or for a shorter period of PEDV in their feces or are protected from PEDV, and furthermore, pigs immunized with PEDV MLV are protected from weight loss and failure to gain weight due to PEDV, and furthermore, PEDV MLV serves as an aid to halt or control the PEDV transmission cycle.

[0096] Also, it should be noted that animals vaccinated with the vaccine of the present invention are immediately safe for human consumption without any significant slaughter withholding period, such as 21 days or less. When provided therapeutically, the vaccine is provided in an effective amount after the signs of actual infection are detected. The dosage for treating an existing infection is about 10-10 6 log 10 TCID 50 or more virus (minimum immunizing dose of vaccine release). The composition can be said to be "pharmacologically" acceptable if the recipient shows tolerance to its administration. Such a composition can be said to be administered in a "therapeutically or prophylactically effective amount" if the amount administered is physiologically significant.

[0097] At least one vaccine or immunogenic composition of the present invention can be administered by any means to achieve the intended purpose using the pharmaceutical composition described herein. For example, the route of administration of such a composition can be by parenteral, oral, oro-nasal, intranasal, intratracheal, topical, subcutaneous, intramuscular, transdermal, intradermal, intraperitoneal, intraocular, and intravenous administration. In one embodiment of the present invention, the composition is administered intramuscularly. Parenteral administration can be by bolus injection or by staged perfusion over time. Any suitable device including a syringe, dropper, needleless injection device, patch, etc. may be used to administer the composition. The route and device selected for use depend on the composition of the adjuvant, the antigen, and the subject, and they are well known to those skilled in the art. Oral or, alternatively, subcutaneous administration is preferred. Oral administration can be direct via water or via feed (solid or liquid sample). When provided in liquid form, the vaccine can be lyophilized and provided as a paste by reconstitution to be added directly to the sample (mixed or placed on top) or otherwise added to water or a liquid sample. Preparation of Vero cells suitable for large-scale virus production The virus of the present invention can be conveniently propagated in the Vero cell stock approved for vaccine production. To produce a safe and approved cell stock, vials of Vero cells were subjected to further subculturing. The cells were passed 4 times through PMEM containing wheat, and a Master Cell Stock (MCS) lot "1834430" was generated. The MCS was tested according to 9 requirements of CFR and EP at PGM - Biological Quality Control; Lincoln, NE. The tests of the MCS were satisfactory for sterility, absence of mycoplasma, and foreign substances. Therefore, the PF - Vero MCS lot "1834430" was considered suitable for submission to the Center for Veterinary Biologics Laboratories (CVB - L) for confirmatory testing.

[0098] The seed origin and passage history are as follows. A comprehensive Vero cell pre-master cell stock was previously frozen. For the preparation of the cell stock, the cells were grown in PMEM (Lincoln product number 00-0779-00) containing 1% bovine serum (product number 00-0710-00, BSE compliant) and 3 mM L-glutamine. They were derived from Vero WCS passage number 136, lot number 071700 MCS+3, 28-Jul-00. A new pre-master cell stock was frozen at passage number 166, which is MCS+33 from the original comprehensive Vero master cell stock. MCS "1833440" was prepared from the pre-master identified as Vero KZO preMaster, and all lot cultures were grown in PMEM containing wheat, 1.0% L-glutamine, and 1.0% fetal bovine serum. On August 14, 2008, cells were seeded into a 150 cm2 T-flask (passage number 167). The flask was incubated for 7 days in 5.0% CO2 at 36.1 °C and then grown (passage number 168). After the flask reached 100% culture density 4 days later, the culture medium was passaged into an 850 cm2 roller bottle (number 169). The roller was incubated at 0.125 - 0.250 rpm, 36.1 °C without using CO2. 4 days later, the final passage of the roller (number 170) was performed. On September 2, 2008, cryopreservation was completed by adding 10.0% fetal bovine serum and 10.0% dimethyl sulfoxide (DMSO ) to the concentrated cell suspension. The vials were labeled with passage level number 170. A total of 231 containers, each containing 4.2 ml, were placed in a controlled rate freezer and transferred into a liquid nitrogen tank for long-term storage in the vapor phase. MCS was prepared without using antibiotics. All reagents used for the preparation of MCS were supplied by Pfizer Global Manufacturing for use in antigen production approved in the domestic and world markets. MCS was prepared by Pfizer’s Master Seed Facility, Lincoln, Nebraska.

[0099] The sterility test was as follows. From September 29, 2008 to October 13, 2008, the Master Cell Stock was tested according to 9CFR(026-ST0) and EP2.6.1. It was found that the MCS was free of bacterial and fungal contamination.

[0100] The mycoplasma test and adventitious agent test were conducted as follows. The MCS was examined according to 9CFR(028-PU0) and EP2.6.7. It was found that the MCS was free of any mycoplasma contamination. The adventitious agent test was completed according to 9CFR113.52 using NL-BT-2 (bovine), Vero, NL-ED-5 (equine), NL-ST-1 (porcine), NL-DK (dog), NL-FK (cat) cells. The MCS was negative for MGG, CPE, and HAd, and negative for BVD, BRSV, BPV, BAV-1, BAV-5, rabies, Reo, BTV, ERV, equine arteritis, PPV, TGE, PAV, HEV, CD, CPV, FPL, and FIP by FA. When the MCS was tested for FIV by ELISA, it was found to be satisfactory.

[0101] The EP adventitious agent test was according to 5.2.4(52-2002). The adventitious agent test using bovine NL-BT-2 and BK (primary), Vero, NL-ED-5 (equine), NL-ST-1 (porcine), MARC MA104, NL-DK (dog), NL-FK (cat) cells was negative for MGG, CPE, HAd, and negative for BVD, BPV, BAV-1, BAV-5, bovine coronavirus, bovine rotavirus, BHV-3, PI3, IBR, BRSV and BEV-1, Reo, BTV, ERV, equine arteritis, PPV, PRV, TGE, HEV, PAV, P. rot A1, rot A2, PRRSV, CD, CPI, CAV-2, measles, C. rot, rabies, CCV, FP, FCV, FVR, FIP, and FeLV by FA. The polypeptides and polynucleotides of the present invention Representative embodiments of the present invention include (a) an ORF1a / 1b polyprotein, a PEDV spike protein (preferably domain 1), an ORF3 protein, an envelope protein, a membrane protein, a nucleocapsid protein, or a fragment(s) of said protein(s) (SEQ ID NOs: 1, 2, 3, 8, 15, 35, 36, 37, 39, and / or 59-77), or a fragment thereof; (b) a complement of any of the sequences of (a); (c) a polynucleotide that hybridizes to the sequence of (a) or (b) under stringent conditions defined as hybridization with filter-bound DNA in 0.5 M NaHPO 4 at 65°C, 7% SDS, 1 mM EDTA, and washing in 0.1× SSC / 0.1% SDS at 68°C; (d) a polynucleotide that is at least 70% identical to the polynucleotide of (a) or (b); (e) a polynucleotide that is at least 80% identical to the polynucleotide of (a) or (b); (f) a polynucleotide that is at least 90% identical to the polynucleotide of (a) or (b); and (g) a polynucleotide that is at least 95% identical to the polynucleotide of (a) or (b), and includes an isolated polynucleotide sequence comprising a polynucleotide selected from the group consisting of. In a preferred embodiment, the polynucleotide comprises a second heterologous polynucleotide sequence.

[0102] The present invention also provides a polypeptide encoded by any of the open reading frames of the genotypes of SEQ ID NOs: 1, 2, 3, 8, 15, 35, 36, 37, 39, 59-78, combinations thereof, or a polypeptide that is at least 90% identical to said polypeptide, its domain, or a fragment thereof, including the option that additional amino acids that are identical in other respects are replaced by conservative substitutions.

[0103] The present invention also provides a polypeptide encoded by any of the open reading frames of the mutant PEDV strain of the present invention, preferably the spike protein, or more preferably the spike protein S1 domain, or a polypeptide that is at least 90% identical to the polypeptide or a fragment thereof, including the option that additional amino acids that are identical in other respects are replaced by conservative substitutions.

[0104] In a preferred embodiment, the polypeptide is expressed from the first 1170 nucleotides of the S1 region of the spike protein of the mutant PEDV strain of the present invention. In a more preferred embodiment, a vaccine based on a PEDV polypeptide is further provided, wherein the antigen is defined by a protein encoded by the open reading frame of SEQ ID NO: 1, 2, 3, 8, 15, 35, 36, 37, 39, 59-77, combinations thereof, or an immunogenic fragment thereof. Further embodiments include that the antigen comprises amino acid sequences encoded by nucleotides of SEQ ID NO: 23-34, 40-58. Further genetic manipulation The polynucleotide and amino acid sequence information provided by the present invention also enables systematic analysis of the structure and function of viral genes and their encoded gene products. Knowledge regarding the polynucleotide encoding the viral gene product of the present invention also enables the use of antisense polynucleotides that recognize and hybridize to the polynucleotide encoding the polypeptide of the present invention or a fragment thereof. Full-length and fragment antisense polynucleotides are useful in this regard. Those skilled in the art will understand that the fragment antisense molecules of the present invention include (i) those that specifically recognize and hybridize to a specific RNA (determined by sequence comparison of the DNA encoding the viral polypeptide of the present invention), and (ii) those that recognize and hybridize to RNA encoding a variant of the encoded protein. Antisense polynucleotides that hybridize to RNA / DNA encoding other PEDV peptides can also be identified by sequence comparison for identifying characteristic or representative sequences of the molecule group, and are further useful for testing antigenic domains in PEDV polypeptides and can also be used to distinguish infection of host animals by non-PEDV members with low relatedness to the family Circoviridae.

[0105] Guidance regarding codon optimization effective for enhancing expression in yeast and E. coli for the constructs of the present invention is generally known to those skilled in the art. antibody Further contemplated by the present invention are anti-PEDV antibodies (e.g., monoclonal and polyclonal antibodies, single-chain antibodies, chimeric antibodies, humanized antibodies, human antibodies, porcine antibodies, and CDR-grafted antibodies comprising compounds containing CDR sequences that specifically recognize the PEDV polypeptides of the present invention. The term "specific for" indicates that the variable region of the antibody of the present invention recognizes and binds only to the PEDV polypeptide (i.e., can distinguish a single PEDV polypeptide from related polypeptides, despite sequence identity, homology, or similarity in the sequences found in the polypeptide group), and this may (optionally) interact with other proteins (e.g., S. aureus protein A, or other antibodies in ELISA technology) via interaction with sequences outside the variable region of the antibody, specifically sequences in the constant region of the Ab molecule. Screening assays for determining the binding specificity of the antibodies of the present invention are well known and routinely performed in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Ma nual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Also contemplated are antibodies that recognize and bind to fragments of the PEDV polypeptides of the present invention, provided that the antibodies are first and most specific for the PEDV polypeptides of the present invention from which the fragments are derived, as defined above.

[0106] For clarity, "antibody" refers to an immunoglobulin molecule capable of binding specifically to an antigen as a result of an immune response to the antigen. Immunoglobulins are serum proteins consisting of "light chain" polypeptide chains and "heavy chain" polypeptide chains having "constant" and "variable" regions, and are classified into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant region. Antibodies include, for example, Fv, Fab’, F(ab’) 2including various forms containing, and capable of existing as single-stranded, synthetic polypeptides containing all or part of one or more antibody single-chain polypeptide sequences. Diagnostic kit The present invention also provides a diagnostic kit. The kit can be useful for distinguishing swine animals naturally infected with field strains of PEDV virus from swine animals vaccinated with any of the PEDV vaccines described herein. The kit can also be useful for detecting animals that may be infected with field strains of PEDV virus before clinical symptoms appear and excluding them from the herd or isolating them from naive or vaccinated animals. The kit includes reagents for analyzing a sample obtained from a swine animal for the presence of antibodies against specific components of a specified PEDV virus. The diagnostic kit of the present invention can include, as a component, one or more peptides from the mutant PEDV strains of the present invention that are present in field strains but not in the vaccine of interest, or vice versa, and the selection of such suitable peptide domains is made possible by extensive amino acid sequencing. As is known in the art, the kit of the present invention can alternatively include, as a component, a peptide provided via a fusion protein. The term "fusion peptide" or "fusion protein" for the purposes of the present invention means a single-stranded polypeptide consisting of at least a part of a PEDV viral protein, preferably ORF1 or ORF3, and a heterologous peptide or protein.

[0107] The following examples are intended to illustrate the above invention and should not be construed as limiting its scope. Those skilled in the art will readily recognize that the examples suggest many other ways in which the present invention can be practiced. It should be understood that while within the scope of the present invention, numerous variations and modifications can be made.

[0108] All inventions disclosed in this specification were made under a joint research agreement (as defined in 35 USC 100(h), 37 CFR 1.9(e)) among the respective assignees of the inventors, Iowa State University, and Zoetis Services LLC, and accordingly, the inventors are entitled to the examination protections provided by 37 CFR 1.104(c)(4)(5).

Example

[0109] Example 1 Sequencing of PEDV S1 (the first 2.2 kb protein of the spike gene) was performed to help determine the genetic relatedness and molecular epidemiology of PEDV in pigs in the United States. Sequencing was performed on 15 PEDV cases at ISU VDL in January 2014. Among them, the PEDV S1 sequences from 10 cases (ISU cases 6 - 15) were similar to each other and also similar to PEDV strains identified in pigs in the United States in 2013 (99.1 - 100% nucleotide identity). Clearly symmetrically, the PEDV S1 sequences from the other 5 cases (ISU cases 1 - 5) had only 93.9 - 94.6% nucleotide identity to PEDV strains previously identified in pigs in the United States in 2013.

[0110] However, these 5 PEDV cases shared 99.6 - 100% nucleotide identity to each other based on the S1 sequences. Phylogenetic analysis based on the S1 sequences showed that the aforementioned 10 PEDV cases (ISU cases 6 - 15) were clustered together with PEDV strains identified in the United States since April 2013. However, the aforementioned 5 PEDV cases (ISU cases 1 - 5) were clustered in a very different manner from PEDV strains previously identified in pigs in the United States (Figure 1). Sequence alignment showed that the S1 sequences of these 5 PEDV cases had several deletions and insertions compared to PEDV viruses previously identified in the United States.

[0111] The S1 gene of this new virus isolate has been determined and is reported herein as SEQ ID NO: 1. Based on currently available data, it is unlikely that this strain is a mutation that evolved from PEDV previously identified in pigs in the United States. The real-time RT-PCR for PEDV provided by ISU VDL targets the nucleocapsid (N) gene. The N-gene is known to be a conserved part of the PEDV genome. So far, the real-time RT-PCR for the PEDV N-gene performed at ISU VDL is thought to readily detect these new PEDVs. The full-length N-gene sequence of the new PEDV was determined and it was similar to PEDV previously identified in the United States.

[0112] Figure 2 shows a phylogenetic tree based on the S1 partial sequence and the full-genome sequence. See the attached materials. In addition to pigs in the United States, US prototype strains were detected in South Korea, Canada, and Mexico, and US mutant INDEL strains were identified in South Korea, Mexico, and Germany. Example 2 At least two genetically different porcine epidemic diarrhea virus (PEDV) strains, the US PEDV prototype strain and the S-INDEL mutant strain, have been identified in the United States of America. The objective of this study was to compare the pathogenicity differences between the US PEDV prototype strain and the S-INDEL mutant strain in conventional neonatal pigs under experimental infection. Fifty PEDV-negative 5-day-old pigs were divided into five groups of 10 each, with a virus titer of 10 4Three US PEDV prototype isolates (IN19338 / 2013-P7, NC35140 / 2013-P7, and NC49469 / 2013-P7), an S-INDEL-mutant isolate (2014020697-P7), and virus-negative culture medium were orally gavaged at 10 ml per pig. All three PEDV prototype isolates tested in this study caused severe intestinal disease in 5-day-old pigs, as indicated by similar pathogenicity regardless of their clade membership, as evidenced by viral shedding in feces and clinical signs such as gross and histopathological lesions. Pigs inoculated with the S-INDEL-mutant isolate had significantly fewer clinical signs, including viral shedding in feces, gross lesions in the small intestine, cecum, and colon, histopathological lesions in the small intestine, and immunohistochemical staining in the ileum, compared to pigs inoculated with the three US PEDV prototype isolates. The US PEDV prototype strains and the S-INDEL mutant strain induced similar viremia levels in inoculated pigs. The complete genomic sequences of the PEDV prototype strains and the S-INDEL mutant strain were determined, but the molecular basis for the differences in virulence among the three PEDV strains will have to be elucidated in the future using reverse genetics approaches. This study provides a strong basis for understanding the molecular mechanisms contributing to PEDV virulence and vaccine attenuation. Results Isolation and sequence comparison of US PEDV Three US PEDV prototype isolates, US / NC35140 / 2013, US / IA49379 / 2013, and US / NC49469 / 2013 were isolated in Vero cells. Typical PEDV cytopathic effects, including syncytium formation and cell detachment, were observed, and virus growth was confirmed by fluorescent immunostaining. All isolates grew efficiently in Vero cells, and the infectious titers ranged from 103 to 106 TCID50 / ml in the first 10 passages.

[0113] The complete genomic sequences of three US PEDV prototype isolates, US / NC35140 / 2013-P7, US / IA49379 / 2013-P7, and US / NC49469 / 2013-P7, were determined and compared with the sequences of the aforementioned US PEDV prototype isolate US / IN19338 / 2013 and the US PEDV S-INDEL-variant isolate US / IL20697 / 2014, and the results are summarized in Table 3. A schematic diagram of the PEDV genome organization and the putative functions of the viral proteins is shown in Figure 12. The prototype isolates US / IN19338 / 2013-P7, US / NC35140 / 2013-P7, US / IA49379 / 2013-P7, and US / NC49469 / 2013-P7 all had genomes that were 28,038 nucleotides in length and had nucleotide (nt) identities of 99.75 - 99.91% at the whole-genome level with respect to each other (differences of 26 - 69 nt). The spike genes of these prototype isolates all had a length of 4,161 nucleotides and had nt identities of 99.54 - 99.88% with respect to each other (differences of 5 - 19 nt). The S-INDEL-variant isolate 2014020697-P7 had a genome that was 28,029 nucleotides in length and had nt identities of 99.08 - 99.22% at the whole-genome level with respect to the four prototype isolates evaluated in this study (differences of 220 - 259 nt). Among them, differences of approximately 64 - 96 nt were located in the ORF1a / 1b region, particularly in the nsp12 and nsp16 regions: However, most of these nt changes on nsp12 and nsp16 were synonymous (silent) substitutions at the amino acid level (Table 3). The significant differences between the US PEDV prototype isolates and the S-INDEL-variant isolates were located in the spike gene (nt identities of 96.25 - 96.37%, differences of 151 - 156 nt), particularly in the S1 portion (nt identities of 93.14 - 93.32%, differences of 148 - 152 nt): Nucleotide substitutions in the S1 portion resulted in putative amino acid changes (Table 3).Compared with the prototype isolate, the S gene of the mutant isolate 2014020697-P7 had three characteristic deletions (a 1-nt deletion of G at position 167, an 11-nt deletion of AGGGTGTCAAT at positions 176 - 186, and a 3-nt deletion of ATA at positions 416 - 418) and one insertion (a 6-nt insertion of CAGGAT between positions 474 and 475).

[0114] Phylogenetic analysis of the PEDV isolates described in this study and 45 PEDV reference sequences is provided in Figure 6. In Figure 6A, a neighbor-joining phylogenetic tree based on the full-genome sequences, the US PEDV prototype strains were clustered together and could be further divided into clade 1 and clade 2, while the US PEDV S-INDEL mutant strains were clustered separately. In Figure 6B, a maximum-likelihood phylogenetic tree based on the full-genome sequences, the US PEDV prototype strains were also clustered into clade 1 and clade 2, while the S-INDEL mutant strains formed a separate subclade within clade 2. In contrast, the phylogenetic clusters in Figure 6C, a neighbor-joining phylogenetic tree based on the S1 sequences, and Figure 6D, a maximum-likelihood phylogenetic tree, were similar. In both Figure 6C and Figure 6D, the US PEDV prototype strains were clustered and could be divided into clade 1 and clade 2, similar to Figure 6A, a neighbor-joining phylogenetic tree based on the full-genome sequences: the US PEDV S-INDEL mutant strains formed a separate clade closely related to several classical PEDV isolates such as Europe / CV777, Korea / SM98, and China / SD-M, which had the same insertion and deletion patterns in the S gene as the US PEDV S-INDEL mutant strains.

[0115] Regarding Figure 6A, 6B which are phylogenetic trees based on the full genome, or Figure 6C, 6D based on S1 Notably, the prototype isolates IN19338 and IA49379 belong to clade 1, and the isolate NC35140 belongs to clade 2. However, the prototype isolate NC49469 belongs to clade 1 in FIGS. 6A and 6B, which are phylogenetic trees based on the whole genome, but belongs to clade 2 in FIGS. 6C and 6D, which are phylogenetic trees based on S1. Three prototype isolates, USA / IN19338 / 2013-P7 (SEQ ID NO: 59), USA / NC35140 / 2013-P7 (SEQ ID NO: 60), and USA / NC49469 / 2013-P7 (SEQ ID NO: 61), as well as one S-INDEL-mutant isolate 2014020697-P7 (SEQ ID NO: 62) were selected to compare their pathogenicity in pigs (Table 4).

[0116] [Table 3]

[0117] [Table 4]

[0118] Clinical evaluation All pigs in G1 (USA / IN19338 / 2013-P7), G2 (USA / NC35140 / 2013-P7), and G3 (USA / NC49469 / 2013-P7) developed soft to watery diarrhea from 1 DPI and persisted until 6 or 7 DPI. Symmetrically, in G4 (IL20697), only one pig showed mild diarrhea with soft stools at 1 DPI. The average diarrhea scores are summarized in FIG. 7A. Overall, as described in the "Materials and Methods" section below, from 0 to 7 DPI, when the diarrhea scores were analyzed, pigs in G1 (P = 0.001) and G3 (P < 0.0001) had significantly higher average diarrhea scores than pigs in G2. Pigs in G1 - G3 inoculated with the prototype PEDV isolates had significantly higher average diarrhea scores than G4 (P < 0.0001) and G5 (negative control, P < 0.0001) inoculated with the PEDV mutant isolate. There was no significant difference in the average diarrhea scores between G4 and G5 (P = 1).

[0119] Throughout the entire experiment, vomiting was not observed in any of the pigs. In G1 - G3 inoculated with the prototype isolate, 1) almost all pigs lost their appetite during the test period and had to be administered enteral nutrition; 2) severe dehydration, rough body hair, and flat or thin flanks were observed in all pigs, and the most severe physical condition was seen at approximately 4 DPI; 3) various degrees of lethargy, including head - down position and recumbent position, were observed from 1 DPI until the end of the test. Symmetrically, in G4 inoculated with the mutant isolate, 1) all pigs had normal appetite; 2) dehydration or lethargy was not observed; 3) at 1 DPI or 2 DPI, 90% of the pigs had mildly flat flanks, but they recovered normally after 4 DPI. All G5 pigs were active and did not show diarrhea, dehydration, lethargy, or anorexia during the test period.

[0120] (-1) to 3 DPI, PEDV - inoculated pigs (G1 - G4) had a significantly lower average daily gain (ADG, P ≤ 0.0001) compared to G5 pigs (negative control ), but there was no significant difference in ADG among G1 - G4 (P - values ranged from 0.089 to 1) (Figure 7B). (-1) to 7 DPI, G1 - G3 (prototype isolate) had a significantly lower ADG than G4 (mutant isolate) (P - values ranged from 0.01 to 0.037), but there was no significant difference in ADG between any of G1, G2, G3, or G4 and G5 (negative control) (P - values ranged from 0.078 to 0.847) (Figure 7B). Virus excretion and distribution From 1 DPI until the end of the experiment, PEDV RNA was detected in rectal swab samples from all pigs of G1 - G3 (prototype isolates). In G4 (mutant isolate), PEDV RNA was detected in rectal swabs from 5 / 10, 8 / 10, 10 / 10, 5 / 5, 5 / 5, 3 / 5, and 2 / 5 pigs at 1, 2, 3, 4, 5, 6, and 7 DPI, respectively. The average genome copy number per milliliter of virus excreted in rectal swabs is summarized in Figure 7C. Pigs in G1 and G2 showed similar levels (P = 0.601) of virus excretion into feces from 1 - 7 DPI, with the number of genome copies / ml ranging from 107.2 - 9.0, corresponding to Ct values of 16 - 22. At 1 - 2 DPI, the highest level of virus excretion into feces was seen in pigs of G3 (about 109 genome copies / ml at Ct 16), and by 7 DPI, virus excretion into feces gradually decreased to about 105.4 genome copies / ml corresponding to a Ct value of 28. Symmetrically, in pigs of G4, virus excretion into feces was seen at about 102.3 genome copies / ml (Ct 31.8) at 1 DPI: virus excretion into feces gradually increased, peaked at 5 DPI (105.4 genome copies / ml at Ct 28.8), and then decreased to about 101.3 genome copies / ml (Ct 36.3) at 7 DPI.

[0121] Statistical analysis showed that significantly higher amounts of viral RNA excretion were seen in rectal swabs from G1 - G3 (prototype isolates) compared to G4 (mutant isolate) (P < 0.0001).

[0122] PEDV RNA was detected in serum samples from all pigs of G1 to G4 necropsied at 3 DPI, and the mean Ct values were 33.2 (G1), 30.5 (G2), 31.9 (G3), and 28.4 (G4). At 3 DPI, there was no significant difference in the mean PEDV genome copy numbers in the sera of G1 to G4 [P values ranged from 0.077 to 0.646] (Figure 7D). At 7 DPI, PEDV RNA was detected in serum samples from 3 to 4 out of 5 pigs of G1 to G4, and the mean Ct values (only for PCR-positive pigs) were 33.5 (G1), 34.2 (G2), 37.4 (G3), and 35.8 (G4). At 7 DPI, there was no significant difference in the mean genome copy number of PEDV in the sera of G4 (mutant isolate) compared with G1 to G3 (prototype isolates) (P values ranged from 0.050 to 0.717) (Figure 7D).

[0123] The viral distribution in tissues is summarized in Table 5. At 3 DPI, regardless of G1, G2, G3, or G4, the mean PEDV RNA concentrations in the ileum, cecum, colon, and mesenteric lymph nodes were higher than those in other tissues within the same inoculation group. When the viral RNA concentrations in each tissue type were compared across the four inoculation groups at 3 DPI, the viral RNA concentrations in the cecum and colon of G4 (mutant isolate) were significantly lower overall than those in the cecum and colon of G1 to G3 (prototype isolates), but the viral RNA concentrations in other tissues of G4 were similar to those in the corresponding tissues of G1 to G3, and the same type of tissues of G1 to G3 had similar levels of viral RNA. The data at 7 DPI generally supported the same conclusions regarding 3 DPI, except that the viral genome copy numbers in the cecum and colon were much less than those in the ileum and mesenteric lymph nodes of G4.

[0124] All rectal swabs, sera, and tissue feeds from G5 (negative control) were negative by real-time RT-PCR for PEDV throughout the test period.

[0125]

Table 5

[0126] Abbreviations: DPI, days post-inoculation; MLN, mesenteric lymph node; PCR, polymerase chain reaction; Ct, cycle threshold * hind limb muscle + The average Ct was that of the PCR-positive pigs (pigs with PCR Ct < 45).

[0127] ‡ Genome copies / ml was the geometric mean of the number of genome copies per milliliter for all pigs (both PCR-positive and PCR-negative pigs). Statistical analysis was performed for the same tissue types in groups G1 - G4, and different letters indicate significant differences. Gross findings At 3 DPI, in most pigs inoculated with the PEDV prototype isolates (G1 - G3), thin, transparent walls distended by gas and / or yellowish fluid were observed in the small intestine, cecum, and colon tissues. Furthermore, almost all pigs in G1 - G3 contained watery contents in the small intestine, cecum, and colon. Symmetrically, only mild thin walls were observed in the small intestine of 3 / 5 pigs inoculated with the PEDV mutant isolate (G4), and no obvious gross lesions were observed in the cecum or colon of G4 pigs. Also, in G4, only 1 / 5 pig contained watery contents in the small intestine, cecum, and colon, and the other 2 pigs had semi-watery contents in the cecum. Statistically, all G1 - G3 (prototype isolates) had significantly higher scores for the contents of the small intestine, cecum, and colon than G4 (mutant isolate) and G5 (negative control) (P values ranged from < 0.0001 to 0.0127) (Fig. 8A). No significant differences were observed among G1–G3 regarding the contents scores of the small intestine, cecum, and colon (P values ranged from 0.3722 to 1) (Fig. 8A). In G4, only the contents score of the cecum was significantly higher than that of G5 (P = 0.003), while the contents scores of the small intestine or colon were not (P values ranged from 0.5259 to 1; Fig. 8A). All prototype isolate-inoculated groups (G1–G3) had significantly higher histopathological lesion scores of the small intestine, cecum, and colon than the mutant isolate-inoculated group (G4) and the negative control group (G5) (P values were 0.0012–0.049, Fig. 8B). There were no significant differences in the lesion scores of the small intestine, cecum, and colon between G1–G3 (P values ranged from 0.1585 to 1) or between G4–G5 [P values ranged from 0.4766 to 1; Fig. 8B].

[0128] At 7 DPI, most pigs in G1–G3 still had a thin-walled and / or gas-distended small intestine, while only about 50% of G1–G3 had a thin-walled and / or gas-distended cecum and colon. All pigs in G1 and G2, and 4 / 5 pigs in G3 contained watery small intestine contents. Approximately 60–100% of pigs in G1–G3 had semi-watery or watery contents in the cecum and colon. In G4, only 1 / 5 pig had a thin-walled small intestine, and none of the pigs had gross lesions in the cecum and colon. In G4, 5 / 5, 1 / 5, and 0 / 5 pigs had semi-watery or watery contents in the small intestine, cecum, and colon, respectively. In G5, 1 / 5 pig had a thin-walled small intestine, cecum, and colon, and 3 / 5 pigs had semi-watery or watery contents in the small intestine, cecum, and colon. Significant differences were observed in the tissue contents scores and tissue lesion scores between some groups (Fig. 8).

[0129] Rectal swabs collected at 3 and 7 DPI were confirmed to be negative for PDCoV, TGEV, and porcine rotavirus (groups A, B, and C), and also negative for hemolytic E. coli and Salmonella spp. Histopathology At either 3 DPI or 7 DPI, no significant microscopic lesions were observed in sections of the stomach, cecum, colon, tonsils, mesenteric lymph nodes, heart, lungs, liver, spleen, and kidneys of all piglets (G1 - G5).

[0130] Severe lesions consistent with viral enteritis (e.g., swelling of villous enterocytes, villous atrophy, disruption of the lamina propria, etc.) were observed in sections of the small intestine (duodenum, jejunum, and ileum) of all pigs in G1 - G3 (prototype isolates) necropsied at 3 DPI and 7 DPI. Mild microscopic lesions consistent with viral enteritis were observed in sections of the small intestine of pigs in G4 (mutant isolate) necropsied at 3 DPI, but the microscopic lesions in sections of the small intestine of pigs in G4 necropsied at 7 DPI were not prominent. No obvious microscopic lesions were observed in sections of the small intestine of pigs in G5 (negative control) at either 3 DPI or 7 DPI. Representative images of H&E - stained ileum sections of pigs in G1 - G5 necropsied at 3 DPI are shown in Figures 9A - 9E.

[0131] Villus height, crypt depth, and the villus height to crypt depth ratio were measured and compared in sections of the small intestine of the five inoculated groups. At 3 DPI, pigs in G1 - G3 (prototype isolates) had, with some exceptions, significantly increased mean villus height, increased mean crypt depth, and a lower mean villus / crypt ratio in the duodenum, proximal jejunum, mid - jejunum, distal jejunum, and ileum compared to G4 (mutant isolate) and G5 (negative control); exceptions included the villus height and villus / crypt ratio in the duodenum of G2 and G4, and the crypt depth in the mid - jejunum of G1 and G4. The mean villus height, crypt depth, and villus / crypt ratio in sections of the small intestine at 3 DPI were overall similar across the three groups G1 - G3 inoculated with the prototype isolate (Figure 10). There was no significant difference in the mean crypt depth of sections of the small intestine at 3 DPI between G4 (mutant isolate) and G5 (negative control), but pigs in G4 had significantly decreased mean villus height and a lower mean villus / crypt ratio in the duodenum, mid - and distal jejunum, and ileum at 3 DPI compared to pigs in G5 (Figure 10).

[0132] At 7 DPI, the mean villus height and villus / crypt ratio of the small intestine sections were overall similar across the three groups G1 - G3 (Figs. 11A - 11D). Pigs in G1 - G3 had, overall, significantly lower mean villus height and lower villus / crypt ratio in the small intestine sections compared to pigs in G4 and G5 at 7 DPI (Figs. 11A, 11C). Interestingly, the mean villus height at 7 DPI was either not significantly different between G4 (mutant isolate) and G5 (negative control) or was significantly higher in G4 than in G5 (Fig. 11A). There was no significant difference in the mean villus / crypt ratio between G4 and G5 in the proximal, middle, and distal jejunum, and ileum at 7 DPI, but a significant difference in the mean villus / crypt ratio in the duodenum between G4 and G5 was observed (Fig. 10C). The comparison of the mean crypt depth at 7 DPI is shown in Fig. 11B. The mean crypt depth was similar between the prototype isolate - inoculated groups G2 and G3 in all small intestine sections, and G2 and G3 had significantly longer crypt depth compared to the negative control group G5. Another prototype isolate - inoculated group G1 had mean crypt depth values between the negative control group G5 and the prototype isolate - inoculated groups G2 and G3. The mutant isolate - inoculated group G4 had significantly increased mean crypt depth in the duodenum, proximal, and distal jejunum compared to the negative control group G5, while G4 had a mean crypt depth similar to G1, G2, and G3 in most of the small intestine sections. Immunohistochemistry (IHC) For 3 DPI, PEDV-specific IHC staining was performed on serial sections of the ileum, cecum, and colon of all five inoculation groups. None of the five pigs in G5 (negative control) were IHC positive in the ileum, cecum, or colon. All five pigs in each of G1 - G4 were IHC positive in the ileum, and the mean IHC scores were 3.9 (G1), 3.7 (G2), 3.8 (G3), and 2.5 (G4). The mean IHC score of the ileum was similar across G1 - G3 but significantly higher than that of G4 (Figure 10D). Regarding IHC staining of the cecum, 5 / 5 (G1), 4 / 5 (G2), 5 / 5 (G3), and 3 / 5 (G4) pigs were positive, and there was no significant difference in the mean IHC scores among G1 - G4 (Figure 10D). In the case of the colon, 5 / 5 (G1), 4 / 5 (G2), 4 / 5 (G3), and 2 / 5 (G4) pigs were positive for IHC staining, but there was no significant difference in the mean IHC scores among G1 - G4 (Figure 10D). Representative PEDV IHC staining images are shown in Figures 9F - 9T.

[0133] For 7 DPI, PEDV-specific IHC staining was performed on serial sections of the ileum. Mild / slight IHC staining was observed in G1 and G2, but no staining was observed in G3, G4, and G5 (Figure 11D). Discussion Array analysis has demonstrated that at least two genetically distinct PEDV strains are circulating in the United States (Vlasova et al., 2014; Wang et al., 2014), which are referred to as the US PEDV prototype strain and the US PEDV S-INDEL mutant strain. The US prototype PEDV can be further divided phylogenetically into clades 1 and 2. In phylogenetic analysis based on the full-genome sequence, the US S-INDEL mutant PEDV clustered separately from clades 1 and 2 within the neighbor-joining phylogenetic tree but formed a separate sub-clade within clade 2 in the maximum-likelihood phylogenetic tree (Figs. 6A, 6B). This suggests that phylogenetic analysis tools and phylogenetic tree construction methods can lead to some differences in the analysis results: Therefore, conclusions should be carefully drawn by clearly indicating the tools and methods used in phylogenetic analysis. Among the US prototype PEDVs, some always belong to clade 1 or clade 2 regardless of whether it is a phylogenetic tree based on the full genome or a phylogenetic tree based on S1, but some (e.g., NC49469 and Minnesota62) belong to clade 1 in the phylogenetic tree based on the full genome but belong to clade 2 in the phylogenetic tree based on S1 (Fig. 6). It is probably because the S1 sequences of NC49469 and Minnesota62 PEDV are more closely related to those of clade 2, while the remaining genomic sequences are more closely related to those of PEDV in clade 1. Our group has isolated various PEDVs belonging to each of the above categories in cell culture, so the pathogenicity of various US prototype and S-INDEL mutant PEDVs can be compared. This is probably because the S1 sequences of NC49469 and Minnesota62 PEDV are more closely related to those of clade 2, while the remaining genomic sequences are more closely related to those of PEDV in clade 1. Our group has isolated various PEDVs belonging to each of the above categories in cell culture, so the pathogenicity of various US prototype and S-INDEL mutant PEDVs can be compared.

[0134] Studies have shown that neonatal piglets are more susceptible to PEDV infection than weaned piglets, and PEDV infection results in higher disease severity in neonates than in weaned piglets (Jung et al., 2015a; Thomas et al., 2015). Therefore, a highly sensitive 5-day-old neonatal piglet model was selected for pathogenicity comparison in this study. Among the three US PEDV prototype isolates (USA / IN19338 / 2013, USA / NC35140 / 2013, and USA / NC49469 / 2013), the average diarrhea score caused by the USA / NC35140 / 2013-P7 isolate was lower than that caused by the USA / IN19338 / 2013-P7 and USA / NC49469 / 2013-P7 isolates, but the three prototype isolates had similar virus excretion, gross lesions, histopathological lesions, and IHC staining. Overall, we concluded that the three US PEDV prototype isolates evaluated in this study had similar pathogenicity in neonatal piglets regardless of their phylogenetic clades. Symmetrically, the data of this study clearly demonstrated that the US PEDV S-INDEL-mutant isolate 2014020697-P7 had significantly reduced clinical signs, virus excretion in feces, gross lesions in the small intestine, cecum, and colon, histopathological lesions in the small intestine, and IHC scores in the ileum compared with the three US PEDV prototype isolates, USA / IN19338 / 2013-P7, USA / NC35140 / 2013-P7, and USA / NC49469 / 2013-P7. Recent experimental studies by other groups have also demonstrated that S-INDEL PEDV has overall lower pathogenicity in 3- to 4-day-old or 1-week-old piglets compared with US prototype strains (Lin et al., 2015a; Yamamoto et al., 2015). However, in the study by Lin et al., it was observed that three litters of piglets inoculated with the US S-INDEL Iowa106 strain showed a mortality rate of zero, while one litter of piglets inoculated with the same virus strain showed a mortality rate of 75% (Lin et al., 2015a).They hypothesized that the health status of sows could directly affect colostrum / milk production and thus influence the infection outcomes of their piglets (Lin et al., 2015a). The prevalence of S-INDEL PEDV observed in the field varies by farm and country. In the United States, infection with the S-INDEL variant OH851 caused only minor clinical signs in farm suckling piglets (Wang et al., 2014). In Germany, two sow farms were infected with an S-INDEL PEDV that had 99.4% nucleotide identity at the whole-genome level to the US S-INDEL variant OH851; however, the severity of clinical signs and mortality in suckling piglets differed significantly between the two farms (Stadler et al., 2015). The factors contributing to the conflicting findings have not yet been clearly identified. However, among S-INDEL PEDVs, the virus source (wild-type or cell culture-adapted virus), inoculation / infection dose, animal / environment conditions, and nucleotide / amino acid diversity may have contributed to the discrepancies observed between various experimental studies and field occurrences. Furthermore, the pathogenicity of PEDV may be age-dependent. Further investigation of the pathogenicity of S-INDEL PEDV variants in weaned pigs, finishing pigs, gilts, and sows is warranted.

[0135] Studies have shown that viremia can occur during the acute phase of infection with US PEDV prototype isolates (Jung et al., 2014; Madson et al., 2016). In this study, we also detected PEDV RNA in serum samples. Furthermore, the PEDV mutant isolate and the three prototype isolates had similar viremia levels under the conditions of this study. PEDV infects villous enterocytes and causes villous atrophy. It is an enteropathogenic coronavirus that causes diarrhea with atrophy and malabsorption. A certain amount of PEDV can enter the bloodstream by a mechanism that is not fully understood. However, PEDV is not thought to replicate actively in the bloodstream, and viremia levels may not necessarily correlate with virulence / pathogenicity. During this study, high levels of PEDV RNA were detected in the small intestine, cecum, colon, and mesenteric lymph nodes, whereas low levels of PEDV RNA were detected in the non-intestinal tissues (tonsils, heart, lung, liver, spleen, kidney, and muscle) of pigs inoculated with either the prototype or S-INDEL mutant PEDV. In previous studies, PEDV viral antigen (US prototype isolate) was detected in the small intestine, mesenteric lymph nodes, and some colon and spleen tissues (Jung et al., 2015b; Jung et al., 2014; Madson et al., 2016), but it was shown that all other non-intestinal tissues, such as the lung, heart, kidney, and liver, were negative for PEDV antigen (Madson et al., 2016). Therefore, detection of PEDV RNA does not necessarily mean that PEDV replicates in all of these non-intestinal tissues. Considering that blood was not drained before collecting each organ, the virus in these tissues may have been due to the undrained blood.

[0136] In this study, PEDV IHC staining was performed only on the ileum, cecum, and colon of inoculated pigs. Among the four groups inoculated with PEDV (three prototype isolates and one mutant isolate), PEDV IHC staining was observed in 100% of the ileum, 60 - 100% of the cecum, and 40 - 100% of the colon at 3 DPI. The mean IHC score of the ileum was significantly lower in pigs inoculated with the mutant isolate than in pigs inoculated with the prototype isolates, which was consistent with the observations on the gross findings and histopathological lesions of the small intestine. The mean IHC scores of the cecum and colon were numerically lower in pigs inoculated with the mutant isolate than in pigs inoculated with the three prototype isolates, but there was no significant difference. However, pigs inoculated with the PEDV mutant isolate had fewer gross changes in the cecum and colon compared to pigs inoculated with the prototype isolates. Therefore, the correlation between the changes in the cecum and colon and the virulence / pathogenicity of PEDV may need to be further elucidated.

[0137] All four groups G1 - G4 (three prototype isolates and one mutant isolate) inoculated with PEDV had significantly shorter villus heights compared to the negative control group G5 at 3 DPI. G1 - G3 (prototype isolates) had significantly shorter villus heights compared to G4 (mutant isolate). These indicate that both the US prototype isolates and mutant PEDV isolates can infect and destroy the villus epithelium of the small intestine, but the US PEDV mutant isolate caused less severe villus atrophy than the prototype isolates. Intestinal crypt epithelial cells play a role in replacing the intestinal cells of the damaged villi. At 3 DPI, there was no significant difference in the average crypt depth of G4 (mutant isolate) compared to G5 (negative control), but the average crypt depth of G1 - G3 (prototype isolates) was significantly longer than that of G4 and G5. These facts may suggest that the mild villus atrophy caused by the US PEDV mutant isolate did not result in significant growth and elongation of intestinal crypts at 3 DPI. However, the intestinal crypts had begun to elongate to some extent to repair the severe villus atrophy in the prototype isolate - inoculated groups G1 - G3. At 7 DPI, since the prototype isolate - inoculated groups had longer average crypt depths than the negative control group, it is suggested that although the crypts continued to replace the damaged villus intestinal cells, the villus epithelium did not recover to normal. Since the average crypt depth of some small intestine sections of G4 (mutant isolate) was significantly longer than that of G5 (negative control) at 7 DPI, it is suggested that in G4, crypt elongation occurred later compared to the prototype isolate - inoculated groups G1 - G3. The proliferated crypts eventually restored the damaged villus intestinal cells seen in G4 at 3 DPI. IHC staining also supported these observations.

[0138] Tests have shown that antibodies against the US PEDV prototype strain and S-INDEL mutant strains cross-react in vitro and can cross-neutralize both strains (Chen et al., 2016; Lin et al., 2015b). In vivo tests (Goede et al., 2015) have shown that female pigs exposed to S-INDEL mutant PEDV infection 7 months prior were able to provide partial protection to neonatal pigs challenged with the US PEDV prototype strain. Another in vivo test (Lin et al., 2015a) demonstrated that 3- to 4-day-old piglets exposed to S-INDEL mutant PEDV were able to provide partial protection against subsequent challenge with US prototype PEDV. The Applicant also owns data demonstrating that both the US PEDV prototype strain and S-INDEL mutant strains can provide homologous and heterologous protection against the two virus strains in a weaned pig model. During this test, the US PEDV S-INDEL mutant strain was shown to be less virulent than the US PEDV prototype strain in neonatal pigs. Collectively, these data suggest that, although further evaluation experiments are needed to determine overall efficacy, the US PEDV S-INDEL mutant strain could potentially be a modified live virus vaccine candidate against PED (see Examples 5 and 6 for additional experiments).

[0139] Significant sequence differences between the US prototype and S-INDEL mutant PEDV are located in the spike gene, particularly the S1 portion. Sequence differences in the spike gene may contribute in part to the difference in virulence between the US prototype and S-INDEL mutant PEDV. Materials and Methods Virus Isolates and Cells The isolation and characterization of the US PEDV prototype isolate US / IN19338 / 2013 and the S-INDEL-mutant isolate US / IL20697 / 2014 have been previously described (Chen et al., 2014, Chen et al., 2016). Three additional US PEDV prototype isolates, US / NC35140 / 2013, US / IA49379 / 2013, and US / NC49469 / 2013, were all obtained from feces of piglets submitted for routine diagnosis and archived at the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) according to previously described virus isolation procedures (Chen et al., 2014) for this study. Isolation, propagation, and titration of PEDV were all performed in Vero cells (ATCC CCL-81) as described (Chen et al., 2014). All PEDV isolates used in this study were confirmed to be negative for porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine rotavirus (A, B, and C), porcine reproductive and respiratory syndrome virus, and porcine circovirus. Virus Sequencing, Comparative Sequence Analysis, and Phylogenetic Analysis As previously described (Chen et al., 2014), the whole-genome sequences of the PEDV isolates described in this study were determined by next-generation sequencing using the Illumina MiSeq platform and assembled using SeqMan Pro version 11.2.1 (DNAstar Inc, Madison, WI). The sequence data of these PEDV isolates were deposited in GenBank under the following accession numbers: US / IN19338 / 2013 [KF650371], US / NC35140 / 2013-P7 [KM975735], US / IA49379 / 2013 [KM975736], US / NC49469 / 2013-P7 [KM975737], and 2014020697-P7 [KT860508].

[0140] The complete genomic sequences and individual gene sequences (nucleotide and amino acid sequences) of all PEDV isolates used in this study were aligned using ClustalX version 2.0 (Larkin et al., 2007) and BioEdit version 7.0.4.1 (Hall, 1999 ) to compare genetic similarities. Phylogenetic analysis was performed using the complete genome and S1 portion (nucleotide 1 to 2205 of the gene according to sequence KF650371) nucleotide sequences of the PEDV isolates described in this specification, as well as worldwide PEDV (50 sequences in total). Phylogenetic trees were constructed using the neighbor-joining method and the maximum likelihood method based on distances in MEGA version 6 (Tamura et al., 2013), respectively. Bootstrap analysis was performed on 1,000 replicated datasets. Experimental design The animal experiment protocol was approved by the Iowa State University Institutional Animal Care and Use Committee (approval number 6-14-7821-S, approved on July 10, 2014). Fifty 5-day-old piglets were purchased from a conventional breeding farm and sent to the facilities of Iowa State University Laboratory Animal Resources. All pigs were intramuscularly injected with Excede® (Zoetis, Florham Park, New Jersey, USA) upon arrival and were confirmed to be negative for PEDV, PDCoV, TGEV, and porcine rotavirus (groups A, B, and C) by virus-specific PCR of rectal swabs and negative for PEDV antibody by virus-specific indirect fluorescent antibody (IFA) assay of serum samples at the ISU VDL. Pigs were grouped by weight and then randomly divided into five groups of 10 each, with one group per room with a hard floor. Animals were fed a mixture of Esbilac (Hampshire, IL) liquid milk replacer and yogurt and allowed free access to water. After 1 day of acclimation (piglets were 6 days old), pigs in groups 1 to 5 (G1 to G5) were orally gavaged with three US PEDV prototype isolates, USA / IN19338 / 2013-P7 (G1), USA / NC35140 / 2013-P7 (G2), USA / NC49469 / 2013-P7 (G3), one US S-INDEL-mutant isolate 2014020697-P7 (G4), or virus-negative culture medium (G5) (10 ml / pig; all viruses were passage 7 in cell culture and had a titer of 104 TCID50 / ml) (Table 4).

[0141] Piglets were evaluated daily for the presence of vomiting and clinical signs of diarrhea, lethargy, and body condition. The severity of diarrhea was scored using the following criteria: 0 = normal, 1 = soft feces (cow dung-like), 2 = liquid containing some solids, 3 = watery without solids. Lethargy levels were classified as normal, mild lethargy (slow movement, head down), moderate lethargy (standing but wanting to lie down), or severe lethargy (recumbent, moribund). Body condition was classified as normal, mild loss (flat flanks), moderate (depression of flanks), or severe (prominence of backbone / ribs).

[0142] Body weights were recorded before inoculation and then at 3 and 7 days post-inoculation (DPI). Average daily gain (ADG) was calculated for pigs from (-1) to 3 DPI and (-1) to 7 DPI. Serum samples were collected at 0, 3, and 7 DPI. Rectal swabs were collected daily from each pig from 0 DPI until necropsy and immediately immersed in 1 ml of PBS after collection. At 3 DPI, 5 pigs were randomly selected from each group for necropsy, and the remaining pigs were necropsied at 7 DPI. Fresh and formalin-fixed samples collected at necropsy included: tonsils, heart, lungs, liver, spleen, kidneys, skeletal muscle from the hindlimbs, stomach, mesenteric lymph nodes, duodenum, proximal jejunum, mid jejunum, distal jejunum, ileum, cecum, and colon. Collection of different intestinal segments was performed as previously described (Madson et al., 2014).

[0143] At necropsy, a veterinary pathologist blinded to the treatment group examined the small intestine, cecum, and colon for gross lesions. Histopathological lesions were classified as normal, thin-walled, and / or inflated by gas. Each thin-walled intestine or organ inflated by gas was counted as 1 point: if both thin-walled and inflated by gas were present, it was counted as 2 points. The contents of the small intestine, cecum , and colon were examined and scored using the following criteria: 0 = normal, 1 = liquid containing some solids (semi-watery), 2 = watery.

[0144] To rule out the possibility of coinfection with other pathogens, rectal swabs collected at 3 DPI and 7 DPI before necropsy were tested for PDCoV, TGEV, and porcine rotavirus (groups A, B, and C) by virus-specific PCR and for hemolytic E. coli and Salmonella spp by routine bacterial culture at the ISU VDL. Virus shedding examined by quantitative real-time RT-PCR based on the PEDV N gene Viral RNA was extracted from rectal swabs, sera, and 10% tissue homogenates as previously described (Chen et al., 2014). The Path-ID (trademark) Multiplex One-Step RT-PCR Kit (Thermo Fisher Scientific) was used, and 5 μl of each RNA template was used in a 25-μl total reaction PCR setup. Primers, probes, and in vitro transcribed RNA used to generate the standard curve for quantitative real-time RT-PCR based on the PEDV N gene have been described (Lowe et al., 2014; Madson et al., 2014; Thomas et al., 2015). Based on the standard curve, the virus concentration was calculated in units of genome copies / ml in the samples tested. The mean cycle threshold (Ct) was calculated based on PCR-positive samples, and the mean virus concentration was calculated based on all pigs within the group (both PCR-positive and negative pigs). Histopathology Tissues from the tonsil, heart, lung, liver, spleen, kidney, mesenteric lymph node, stomach, duodenum, proximal jejunum, mid jejunum, distal jejunum, ileum, cecum, and colon were fixed in 10% formalin, embedded, sectioned, stained with hematoxylin and eosin (H&E), and examined by a veterinary pathologist blinded to the identity and treatment group of the individual animals. Using a computer image system according to the previously described procedure (Madson et al., 2014), villus length and crypt depth were measured from three representative villi and crypts of the duodenum, proximal jejunum, mid jejunum, distal jejunum, and ileum. The ratio of villus height to crypt depth (villus / crypt) for each tissue was calculated as the quotient of the mean villus length divided by the mean crypt depth. Immunohistochemistry Serial sections of the ileum, cecum, and colon at necropsy at 3 DPI were evaluated for PEDV antigen by immunohistochemistry (IHC) using a previously described (Madson et al., 2014) PEDV-specific monoclonal antibody (BioNote, Hwaseong-si, Gyeonggi-do, Korea). At necropsy at 7 DPI, IHC staining was performed only on serial sections of the ileum. Detection of IHC antigen was semi-quantitatively scored as previously described (Chen et al., 2015) using the following criteria: 0 = no staining; 1 = positive staining in approximately 1 - 10% of enterocytes; 2 = positive staining in approximately 10% - 25% of enterocytes; 3 = positive staining in approximately 25% - 50% of enterocytes; 4 = positive staining in approximately 50% - 100% of enterocytes. Statistical Analysis A generalized linear mixed (GLIMMIX) model was used for all statistical comparisons using Statistical Analysis System (SAS) version 9.3 (SAS Institute, Cary, NC). A P-value < 0.05 was defined as statistically significant. P-values for overall viral excretion levels in feces [Log10 (genome copies / ml)] were evaluated between 0 - 7 DPI of treatment using DPI and treatment as interaction variables and using a similar approach for the analysis of diarrhea scores. References Boniotti, M.B., Papetti, A., Lavazza, A., Alborali, G., Sozzi, E., Chiapponi, C., Faccini, S., Bonilauri, P., Cordioli, P. & Marthaler, D. (2 016). Porcine Epidemic Diarrhea Virus and Discovery of a Recombinant Swine Enteric Coronavirus, Italy. Emerg Infect Dis 22, 83 - 87. Chen, Q., Gauger, P., Stafne, M., Thomas, J., Arruda, P., Burrough, E., Madson, D., Brodie, J., Magstadt, D., Derscheid, R., Welch, M. & Zhang, J. (2015). Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets. Virology 482, 51-59. Chen, Q., Li, G., Stasko, J., Thomas, J.T., Stensland, W.R., Pillatzki, A.E., Gauger, P.C., Schwartz, K.J., Madson, D., Yoon, K.J., Stevenson, G.W., Burrough, E.R., Harmon, K.M., Main, R.G. & Zhang, J. (2014). Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States. J Clin Microbiol 52, 234-243. Chen, Q., Thomas, J.T., Gimenez-Lirola, L.G., Hardham, J.M., Gao, Q., Gerber, P.F., Opriessnig, T., Zheng, Y., Li, G., Gauger, P.C., Madson, D.M., Magstadt, D. & Zhang, J. (2016). Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains. BMC Vet Res (in revision). 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Thomas, J. T., Chen, Q., Gauger, P. C., Gimenez-Lirola, L. G., Sinha, A., Harmon, K. M., Madson, D. M., Burrough, E. R., Magstadt, D. R., Salzbrenner, H. M., Welch, M. W., Yoon, K. J., Zimmerman, J. J. & Zhang, J. (2015). Effect of Porcine Epidemic Diarrhea Virus Infectious Doses on Infection Outcomes in Naive Conventional Neonatal and Weaned Pigs. PLoS One 10, e0139266. Van Diep, N., Norimine, J., Sueyoshi, M., Lan, N. T., Hirai, T. & Yamaguchi, R. (2015). US-like isolates of porcine epidemic diarrhea virus from Japanese outbreaks between 2013 and 2014. Springerplus 4, 756. Vlasova,A.N.,Marthaler,D.,Wang,Q.,Culhane,M.R.,Rossow,K.D.,Rovira,A.,Collins,J.&Saif,L.J.(2014).Distinct characteristics and complex evolution of PEDV strains,North America,May 2013-February 2014.Emerg Infect Dis 20,1620-1628. Wang,L.,Byrum,B.&Zhang,Y.(2014).New variant of porcine epidemic diarrhea virus,United States,2014.Emerg Infect Dis 20,917-919. Yamamoto,R.,Soma,J.,Nakanishi,M.,Yamaguchi,R.&Niinuma,S.(2015).Isolation and experimental inoculation of an S INDEL strain of porcine epidemic diarrhea virus in Japan.Res Vet Sci 103,103-106. Example 3 At least two genetically distinct porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States of America (US PEDV prototype strain and S-INDEL mutant strain). Current serological assays provided by veterinary diagnostic laboratories for the detection of PEDV-specific antibodies are based on the US PEDV prototype strain. The objectives of this study were to 1) isolate the US PEDV S-INDEL mutant strain in cell culture, 2) produce antisera against the US PEDV prototype strain and S-INDEL mutant strain by experimentally infecting weaned pigs, and 3) determine whether various PEDV serological assays can detect antibodies against the US PEDV S-INDEL mutant strain or vice versa. In this study, the US PEDV S-INDEL mutant strain was isolated in cell culture. Three groups of PED Three-week-old V-negative pigs (5 pigs per group) were orally inoculated with a US PEDV prototype isolate (previously isolated in our laboratory), an S-INDEL mutant isolate, or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21, and 28 days post-inoculation were evaluated by the following PEDV serological assays: 1) an indirect fluorescent antibody (IFA) assay using the prototype strain and the S-INDEL mutant strain as indicator viruses, 2) a virus neutralization (VN) test against the prototype and S-INDEL mutant viruses, 3) an ELISA based on the whole virus of the PEDV prototype strain, 4) an ELISA based on S1 of the PEDV prototype strain, and 5) an ELISA based on S1 of the PEDV S-INDEL mutant strain. Positive antisera against the prototype strain reacted with and neutralized both the prototype and S-INDEL mutant viruses, and positive antisera against the S-INDEL mutant strain also reacted with and neutralized the prototype and S-INDEL mutant viruses as examined by the IFA antibody assay and VN test. Antibodies against the two PEDV strains could be detected by all three ELISAs, but the detection rates were somewhat different. The Applicant demonstrates that antibodies against the US PEDV prototype strain and the S-INDEL mutant strain cross-react and cross-neutralize both strains in vitro. Current serological assays based on the US PEDV prototype strain can detect antibodies against both US PEDV strains.

[0145] Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), was first recorded in the UK in the early 1970s and has since spread to European and Asian countries [1]. In North America, PEDV was first detected in the United States (US) in April 2013 [2], and subsequently reported in Canada [3] and Mexico [4]. PEDV is an enveloped, single-stranded positive-sense RNA virus belonging to the order Nidovirales, family Coronaviridae, subfamily Coronavirinae, genus Alphacoronavirus [5]. The PEDV genome is approximately 28 kb in length and contains open reading frames (ORFs) encoding the replicase polyproteins ORF1a and ORF1b, as well as four structural proteins [spike (S), envelope (E), membrane (M), and nucleocapsid (N)] and one non-structural protein NS3B (encoded by ORF3) [1].

[0146] In the United States, during the first PED epidemic, a highly virulent PEDV strain (US PEDV prototype strain) was identified [2, 6, 7]. Recently, based on field observations, a PEDV variant strain with insertions and deletions (INDELs) in the spike gene compared to the US prototype strain, and with mild clinical signs, was identified in pigs in the United States [8]. This US PEDV variant strain is also known as the S INDEL strain [4] and forms a distinct phylogenetic cluster compared to the US PEDV prototype strain [4, 8, 9]. During the isolation of PEDV in cell culture, one PEDV isolate (PC177) with a 197-aa deletion in the N-terminal S protein was discovered: However, this PEDV isolate still clustered phylogenetically with the US PEDV prototype strain and was not considered as one of the S-INDEL variant strains

[10] . Marthaler et al.

[11] reported a "third" strain of PEDV (Minnesota188) in pigs in the United States with a 6-nucleotide deletion (2-amino acid deletion) in the spike gene (different from the US S-INDEL variant strain). However, since PEDV Minnesota188 was genetically very closely related to the US PEDV prototype strain, there is some debate as to whether it should be referred to as the "third" strain of PEDV in the United States. PEDV PC177 and Minnesota188 are probably mutations of the US PEDV prototype strain. Thus, at least two genetically distinct PEDV strains, the US PEDV prototype strain and the S-INDEL variant strain, are currently circulating in the United States.

[0147] The US PEDV prototype strain was successfully isolated and propagated in cell culture for several groups [7, 10, 12, 13]. A number of serological assays, including indirect fluorescent antibody (IFA) assay, virus neutralization (VN) test, whole-virus-based enzyme-linked immunosorbent assay (ELISA), recombinant S1 protein-based ELISA, and recombinant nucleocapsid protein-based ELISA, have been developed for the detection of PEDV-specific antibodies [14-18]. All of these serological assays are based on the US PEDV prototype strain.

[0148] In this study, the applicant isolated the US PEDV S-INDEL mutant strain in cell culture. The US PEDV prototype strain and the newly isolated US PEDV S-INDEL mutant strain were each experimentally inoculated into pigs to produce strain-specific antisera. Subsequently, the produced porcine antisera were subjected to in vitro evaluation for serological cross-reactivity and cross-neutralization between the two strains. Specifically, 1) perform a PEDV IFA antibody assay (using the prototype strain and the S-INDEL mutant strain as the respective indicator viruses) and ELISA (ELISA based on the whole virus of the PEDV prototype strain, ELISA based on PEDV prototype strain S1, and ELISA based on PEDV S-INDEL mutant strain S1) to evaluate the antibody cross-reactivity of the two US strains, and 2) perform a VN test using the prototype strain and the S-INDEL mutant strain as the indicator viruses to evaluate the in vitro cross-neutralization of the two US strains. Materials and Methods Isolation of the US PEDV S-INDEL Mutant Strain in Cell Culture To achieve virus isolation in Vero cells (ATCC CCL-81) according to the previously described procedure [7], 68 clinical samples (27 fecal swabs, 24 feces, 13 small intestines, and 4 oral fluids) that were positive by real-time RT-PCR based on the PEDV N gene [17, 19] and confirmed to be positive for the US PEDV S-INDEL mutant strain but negative for the US prototype strain by S1 sequencing were selected at the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL).

[0149] Of the 68 clinical samples that were positive for the US PEDV S-INDEL mutant strain, one small intestine homogenate from pigs in Illinois (cycle threshold (Ct) of real-time RT-PCR based on the PEDV N gene was 16.1) [17, 19] was orally gavaged (10 ml per pig) to three 3-week-old PEDV-naive weaned pigs. The homogenate used for inoculation was confirmed to be negative for transmissible gastroenteritis virus (TGEV), porcine rotavirus A, B, C groups, and porcine deltacoronavirus (PDCoV) by virus-specific RT-PCR at the ISU VDL. Rectal swabs and feces were collected twice a day from each inoculated pig and examined by real-time RT-PCR for PEDV on the same day. Pigs were euthanized when the RT-PCR Ct value of the rectal swab reached <15 and necropsied within 24 hours. Small intestine tissue and cecal contents were collected to achieve virus isolation in cell culture as previously described [7]. This animal experiment was conducted according to the procedures approved by the Iowa State University Institutional Animal Care and Use Committee (IACUC, approval number 3-14-7766-S).

[0150] As previously described, the whole genome sequence of the US / IL20697 / 2014, a cell culture isolate of the US PEDV S-INDEL mutant strain obtained in this study, was determined by next-generation sequencing (NGS) technology using the Illumina MiSeq platform [7]. The sequences of the PEDV S1 portion of the isolate US / IL20697 / 2014 and the clinical sample from which the virus isolate was derived were determined by Sanger sequencing according to the previously described procedures [7]. Production of antisera against US prototype and S-INDEL mutant PEDV By real-time RT-PCR of rectal swabs and by Fifteen 3-week-old pigs confirmed to be negative for PEDV were randomly divided into three groups, with five pigs per group and similar average weights per group. After acclimation for 3 days, the pigs in the three groups were orally gavaged with the US PEDV prototype cell culture isolate, US / IN19338 / 2013-P7 (Pro group) (SEQ ID NO: 59) [7], the US PEDV S-INDEL mutant cell culture isolate, US / IL20697 / 2014-P7 (SEQ ID NO: 62) (Var group), and virus-negative culture medium (Neg group) at a virus titer of 104 TCID50 / ml and 10 ml per pig. Rectal swabs were collected daily from all pigs at 0 - 7 DPI, and then at 10, 14, 21, and 28 DPI and examined by quantitative real-time RT-PCR based on the PEDV N gene

[20] to confirm infection. Serum samples were collected from all pigs at 0, 7, 14, 21, and 28 days post-ingestion (DPI) for evaluation of cross-reactivity and cross-neutralization. This animal experiment was conducted according to the procedures approved by the Iowa State University IACUC committee (approval number 6-14-7809-S).

[0151] Twenty-five serum samples collected at 0, 7, 14, 21, and 28 DPI from the Pro group (Pro antiserum), twenty-five serum samples collected from the Var group (Var antiserum), and twenty-five serum samples collected from the Neg group (Neg antiserum) were examined by various serological assays during this study. Additionally, one porcine antiserum against the European PEDV CV777 strain, one porcine antiserum against the TGEV Purdue strain, one porcine antiserum against porcine hemagglutinating encephalomyelitis virus (PHEV), one porcine antiserum against porcine respiratory coronavirus (PRCV), and one porcine antiserum against PDCoV were included in this study for evaluation. Antisera against the PEDV CV777, TGEV Purdue, and PHEV strains were purchased from the National Veterinary Service Laboratory, Ames, IA. The antisera against PRCV and PDCoV were positive control sera obtained from the ISU VDL. Indirect fluorescent antibody (IFA) assay Eighty serum samples were tested by immunofluorescence assay (Pro IFA) based on the PEDV prototype strain and immunofluorescence assay (Var IFA) based on the S-INDEL mutant strain according to the previously described procedure

[20] . The US / IN19338 / 2013, a PEDV prototype isolate, was used as the indicator virus in the Pro IFA assay, and the US / IL20697 / 2014, an S-INDEL-mutant isolate, was used as the indicator virus in the Var IFA assay. A positive signal at a serum dilution rate of 1:40 or higher was considered IFA antibody positive. PEDV ELISA for antibody detection For the detection of PEDV-specific IgG antibodies, an indirect ELISA (ProWV ELISA) based on the whole virus of the US PEDV prototype strain was developed and validated at the ISU VDL [15, 16]. All serum samples were tested by this ProWV ELISA according to the procedure described in detail

[20] . A sample-to-positive (S / P) ratio > 0.8 was considered antibody positive, an S / P ratio of 0.6 - 0.8 was considered a suspect case, and an S / P ratio < 0.6 was considered negative.

[0152] To test all serum samples in this study, an indirect ELISA (ProS1 ELISA) based on the S1 of the US PEDV prototype strain was used according to the previously described procedure

[14] . An S / P ratio > 0.2 was considered antibody positive, an S / P ratio of 0.14 - 0.2 was considered a suspect case, and an S / P ratio < 0.14 was considered negative.

[0153] An indirect ELISA (VarS1 ELISA) based on the S1 of the US PEDV S-INDEL mutant strain was developed for this study. The region encoding the S1 portion (aa 1 - 735) of the US PEDV S-INDEL mutant strain was codon-optimized, with a 5' Kozak sequence, 5' eukaryotic The object signal sequence and a 3’ 6×-His tag were added and synthesized by GeneArt® Gene Synthesis (Thermo Fisher Scientific, Waltham, MA, USA). The resulting 2,358 base pair DNA fragment was cloned into the eukaryotic expression vector (pZOE15) owned exclusively by Zoetis. The authenticity and orientation of the inserted fragment in the recombinant plasmid were confirmed by sequencing. The recombinant plasmid was transiently transfected into human embryonic kidney (HEK) 293 cells using the PEI transfection method owned exclusively by Zoetis. Seven days after transfection, the culture supernatant was collected and filter sterilized. The recombinant protein was purified by the Ni-NTA Purification System (Thermo Fisher Scientific). The optimal antigen concentration and optimal serum dilution ratio for VarS1 ELISA were determined using checkerboard titration. A 96-well polystyrene microtiter plate (Nunc®, Thermo Fisher Scientific) was coated with the PEDV mutant S1 protein (100 μl per well) and incubated overnight at 4°C. After washing 5 times with PBS, the plate was blocked with PBS containing 1% bovine serum albumin (Jackson ImmunoResearch Inc., West Grove, PA, USA) for 2 hours at 25°C (300 μl / well). The plate was dried at 37°C for 4 hours and then stored at 4°C in a sealed bag containing a desiccant pack until use. Serum samples were diluted 1:50 and added to the coated plate (100 μl / well). The plate was incubated at 25°C for 1 hour and then washed 5 times with PBS. Then, 100 μl of peroxidase-conjugated goat anti-pig IgG (H+L) (Jackson ImmunoResearch Inc., West Grove, PA, USA) was added at a dilution ratio of 1:25,000 and the plate was incubated at 25°C for 1 hour. After the washing step, 100 μl of tetramethylbenzidine-hydrogen peroxide substrate (TMB, Dako North America Inc., Carpinteria, CA, USA) was added.The plate was incubated at room temperature for 5 minutes, and the reaction was stopped by adding 50 μl of stop solution (1 M sulfuric acid). The reaction was measured as the optical density (OD) at 450 nm using a commercially available software-operated (Biotek® Instruments Inc., Winooski, VT, USA) ELISA plate reader. The serum antibody response was expressed as the sample-to-positive (S / P) ratio calculated as follows: S / P ratio = (sample OD - mean negative control OD) / (mean positive control OD - mean negative control OD). The PEDV VarS1 ELISA was validated using 29 field serum samples collected from farms where exposure to the US PEDV S-INDEL mutant strain (serum samples were collected from 29 weaned pigs 1 month after being found positive for the S-INDEL mutant strain by PCR) was confirmed, and 20 PEDV-negative field serum samples. An S / P ratio of >0.3 was considered antibody positive, 0.2 - 0.3 was considered a suspect case, and <0.2 was considered negative. Virus neutralization (VN) test Serum samples were tested by VN based on the US PEDV prototype strain (Pro VN) and VN based on the US PEDV S-INDEL mutant strain (Var VN) according to previously described procedures

[20] . The US / IN19338 / 2013, a PEDV prototype isolate, was used as the indicator virus in the Pro VN assay, and the US / IL20697 / 2014, an S-INDEL - mutant isolate, was used as the indicator virus in the Var VN assay. The reciprocal of the highest serum dilution rate that resulted in a >90% reduction in staining compared to the negative serum control was defined as the VN titer of the serum sample. A VN titer of ≧8 was considered positive. Statistical analysis The Log2(IFA titer / 10) of the Pro antisera and Var antisera tested by Pro IFA and Var IFA were tested in a generalized linear mixed (GLIMMIX). The number of days post - ingestion and the antigen were used as independent variables, and the pig ID and the interaction between the pig ID and the antigen were set as random effects. The Pro tested by Pro VN and Var VN The Log2(VN titer) of the antiserum and Var antiserum was also analyzed similarly. For ELISA analysis, ELISA antigen, pig ID, and pig DPI were used as independent variables. All statistical analyses were performed using Statistical Analysis System (SAS) version 9.3 (SAS institute, Cary, NC, USA), and a P-value of <0.05 was considered significant. Results Isolation of US PEDV S-INDEL mutant strains in cell culture First, attempts were made to isolate the virus from 68 clinical samples that were positive for US PEDV S-INDEL mutant strains received at the ISU VDL, but the isolation of the virus in cell culture ended in failure. Subsequently, three 3-week-old pigs were inoculated with an intestinal homogenate positive for the PEDV S-INDEL mutant strain. Rectal swabs from the pigs had PEDV RT-PCR Ct <15 at 2 DPI, and the pigs were euthanized and necropsied at 3 DPI. Rectal swabs from the other two pigs had PEDV RT-PCR Ct <15 at 3 DPI, and both pigs were euthanized and necropsied at 4 DPI. Small intestinal tissues and cecal contents collected at necropsy were used for virus isolation in Vero cells. The US PEDV S-INDEL mutant strain was successfully isolated from small intestinal homogenates and cecal contents collected from all three pigs. Typical cytopathic effects of PEDV, including syncytium formation and cell detachment, were observed, and virus growth was confirmed by fluorescence immunostaining using the PEDV-specific monoclonal antibody SD6-29.

[0154] One US PEDV S-INDEL mutant isolate designated as US / IL20697 / 2014 was selected for further propagation and characterization. When this isolate was passaged continuously in Vero cells, the infectious titer was in the range of 103-105 TCID50 / ml in the first 10 passages. The complete genomic sequence of the 5th passage of isolate 2014020697-P5, lineage 1 (SEQ ID NO: 8) had 99.3-99.9% nucleotide identity to other US PEDV S-INDEL mutant sequences available in GenBank. The S1 sequence of US / IL20697 / 2014 cell culture isolate P5 had 99.8% nucleotide identity to the original intestinal homogenate from which the virus isolate was derived (only 4 nucleotide differences). The US / IL20697 / 2014 isolate was tested at the ISU VDL and was confirmed to be negative for TGEV, PRCV, PDCoV, porcine rotavirus A, B, C, influenza A virus, porcine reproductive and respiratory syndrome virus, and porcine circovirus 2 by virus-specific PCR. Preparation of antisera against US prototype and S-INDEL mutant PEDV As confirmed by rectal swab PCR testing, the US PEDV prototype isolate, US / IN19338 / 2013-P7 (SEQ ID NO: 59), and the S-INDEL mutant isolate, 2014020697-P7 (SEQ ID NO: 62), were established in all inoculated pigs. When examined by real-time RT-PCR for PEDV, in the prototype group, 4 / 5, 5 / 5, 5 / 5, 5 / 5, 5 / 5, 5 / 5, and 3 / 5 pigs shed virus in rectal swabs at 2, 4, 7, 10, 14, 21, and 28 DPI, respectively. In the S-INDEL mutant group, 3 / 5, 5 / 5, 5 / 5, 5 / 5, 4 / 5, 3 / 5, and 1 / 5 pigs shed virus in rectal swabs at 2, 4, 7, 10, 14, 21, and 28 DPI, respectively. Rectal swabs from negative control pigs remained PEDV PCR negative throughout the test period. Overall, 25 antisera were collected from prototype strain-inoculated pigs (Pro antisera) at 0, 7, 14, 21, and 28 DPI, 25 antisera were collected from mutant strain-inoculated pigs (Var antisera), and 25 antisera were collected from the negative control group (Neg antisera). Evaluation of cross-reactivity of antibodies against US PEDV prototype and S-INDEL mutant strains by PEDV IFA antibody assay As shown in Figure 13, Pro antisera were antibody negative (0 / 5) at 0 and 7 DPI in the IFA antibody assay based on the prototype strain (Pro IFA), and 100% positive (5 / 5) at 14, 21, and 28 DPI. IFA based on the mutant strain (Var IFA) showed similar results for Pro antisera, except that sera collected on day 14 DPI were negative in the Var IFA assay. When antibody titers were compared, positive Pro antisera reacted better overall against the Pro IFA assay than against the Var IFA assay, with an average titer 1.4 log2 higher (Figure 13).

[0155] The Var antiserum was negative at 0 and 7 DPI (0 / 5) in both the Pro IFA and Var IFA antibody assays and 100% positive at 14, 21, and 28 DPI (5 / 5). When comparing the antibody titers, the positive Var antiserum reacted similarly in both the Pro IFA and Var IFA assays, and on average the difference in titers was less than 0.1 log2 (Figure 13).

[0156] The antiserum collected from the negative control group (Neg antiserum) was antibody negative in both the PEDV Pro IFA and Var IFA assays throughout the test. The porcine antiserum against the European PEDV CV777 strain had similar antibody titers in the Pro IFA assay (titer 320) and the Var IFA assay (titer 160). The antisera against TGEV Purdue, PHEV, PDCoV, and PRCV viruses were all negative in both the PEDV Pro IFA and Var IFA assays. Evaluation of the cross-reactivity of antibodies against US PEDV prototype strains and S-INDEL mutant strains by various PEDV ELISAs As shown in Figure 14, the Pro antisera collected at 0 and 7 DPI were all antibody negative in the ProWV ELISA, ProS1 ELISA, and VarS1 ELISA. For the Pro antisera collected at 14 DPI, in the ProWV ELISA, 2 sera were positive and 3 were in the range of suspect cases; in the ProS1 ELISA, 3 were positive and 1 was a suspect case; in the VarS1 ELISA, 2 were positive and 1 was a suspect case. The Pro antisera collected at 21 and 28 DPI were all positive in the three ELISAs. When comparing the total numbers of Pro antisera that were positive at 14, 21, and 28 DPI by each ELISA, there was no significant difference among the three ELISAs for detecting antibodies against the US PEDV prototype strain.

[0157] The Var antisera collected at 0 and 7 DPI were all negative in all three ELISAs, with the exception that one serum at 7 DPI was in the range of suspect cases in the ProS1 ELISA (Figure 14). The Var antisera collected at 14, 21, and 28 DPI had variable positive, suspect, and negative results in the three ELISAs (Figure 14). Overall for the Var antisera, the ProWV ELISA detected 14 sera as antibody positive, 1 as suspect, and 10 as negative; the ProS1 ELISA detected 8 sera as positive, 5 as suspect, and 12 as negative; and the VarS1 ELISA detected 12 sera as positive, 3 as suspect, and 10 as negative. When comparing the total number of Var antisera that were positive at 14, 21, and 28 DPI by each ELISA, the ProWV ELISA was significantly better than the ProS1 ELISA for detecting antibodies against the US PEDV S-INDEL mutant strain (p = 0.0079). However, there was no significant difference between the ProWV ELISA and the VarS1 ELISA (p = 0.3643) or between the ProS1 ELISA and the VarS1 ELISA (p = 0.0723) for detecting antibodies against the US PEDV S-INDEL mutant strain.

[0158] The antisera collected from the negative control group (Neg antisera) were antibody negative in all three PEDV ELISAs throughout the test period from 0 to 28 DPI. The porcine antisera against the European PEDV CV777 strain were antibody positive in all three PEDV ELISAs. TGEV The antisera against Purdue, PHEV, PDCoV, and PRCV viruses were all negative in the three PEDV ELISAs. Evaluation of cross-neutralization of antibodies against US PEDV prototype and S-INDEL mutant strains by virus neutralization test As shown in Figure 15, regardless of the test by the Pro VN or Var VN assay, VN antibodies were detected as early as 7 DPI in the sera of most pigs inoculated with either the prototype strain or the S-INDEL mutant strain. Serum samples collected at 14, 21, and 28 DPI from all pigs inoculated with the PEDV prototype strain or the S-INDEL mutant strain were VN antibody positive in both the Pro VN and Var VN assays.

[0159] The positive Pro antiserum had similar VN antibody titers in the Pro VN and Var VN assays, and there was no significant difference between the two assays. The positive Var antiserum showed similar VN antibody titers in the Pro VN and Var VN assays. Overall, there was no significant difference between the two assays (p = 0.42), but the mean VN antibody titers of the Var antiserum at 21 and 28 DPI were slightly higher in the Var VN assay than in the Pro VN assay (Figure 15).

[0160] The VN antibody titers of the positive Pro antiserum tested by the homologous Pro VN assay were, on average, 0.8 log2 higher than those of the positive Var antiserum tested by the homologous Var VN assay (Figure 15).

[0161] The antiserum collected from the negative control group (Neg antiserum) was antibody negative in both the Pro VN and Var VN assays throughout the test period from 0 to 28 DPI. The porcine antiserum against the European PEDV CV777 strain was antibody positive in the Pro VN assay (titer 64) and the Var VN assay (titer 16). The antisera against TGEV Purdue, PHEV, PDCoV, and PRCV viruses were all negative in both the PEDV Pro VN and Var VN assays. Discussion The applicant has previously isolated the US PEDV prototype strain in Vero cells [7]. To obtain cell culture isolates of the US PEDV S-INDEL mutant strain for the production of strain-specific antisera for evaluation, initially, virus isolation was attempted in Vero cells using 68 PEDV S-INDEL mutant strain-positive clinical samples submitted to the ISU VDL. However, attempts to isolate the S-INDEL mutant virus in cell culture from these samples were unsuccessful. This may be due to multiple factors such as low virus concentration in the samples, cytotoxicity of some samples, and various storage conditions of the clinical samples after collection. Next, one intestinal homogenate containing S-INDEL mutant PEDV among the 68 clinical samples was inoculated into pigs to generate fresher material with a sufficient virus load for attempts at virus isolation in cell culture. Using this approach, the US S-INDEL mutant PEDV was successfully isolated in Vero cells. It is speculated that high virus concentration in the samples and immediate attempts at virus isolation on fresh samples are the keys to success in virus isolation in cell culture. In the case of other viruses that are difficult to directly isolate their viruses in cell culture from clinical samples of naturally infected animals, the approach described in this study, i.e., amplifying the virus in the host animal to obtain fresh samples containing high concentrations of virus for attempting virus isolation in cell culture, may be considered.

[0162] Some field serum samples collected from swine farms were submitted to the ISU VDL for PEDV antibody detection. However, due to the lack of an obvious exposure history in those cases and the possibility of being infected with multiple pathogens or one or more PEDV strains, those field serum samples were not ideal for evaluating the serological cross-reactivity of different PEDV strains. Therefore, in the tests of the present invention, for the evaluation of cross-reactivity by various serological assays antiserum against the US PEDV prototype strain and S-INDEL mutant strain was produced in weaned pigs under strict experimental conditions.

[0163] When examined by IFA antibody assay, the positive antiserum against the prototype strain reacted with both the prototype and S-INDEL mutant viruses, and the positive antiserum against the S-INDEL mutant strain also reacted with both the prototype and S-INDEL mutant viruses. When considering the antibody titers, the antibody against the prototype strain reacted better with the Pro IFA assay than with the Var IFA assay, whereas the antibody against the S-INDEL mutant strain reacted similarly with both the Var IFA and Pro IFA assays. Therefore, the IFA antibody assay based on the US PEDV prototype strain of the present invention provided by the veterinary diagnostic laboratory can be used to reliably detect antibodies against both US PEDV strains.

[0164] ProWV ELISA and ProS1 ELISA have been previously developed and validated for detecting PEDV-specific antibodies [14-16]. The VarS1 ELISA for PEDV was developed in this study. However, this VarS1 ELISA was only validated using a limited number of field antisera against US PEDV S-INDEL mutant strains before testing the antisera prepared experimentally in this study. Further validation of this VarS1 ELISA using a large number of serum samples was required to determine the performance of this assay. All three PEDV ELISAs reacted with Pro antisera and Var antisera. The three ELISAs detected the similarity of Pro antisera. However, the ProS1 ELISA used in this study was felt to be less efficient than the ProWV ELISA and VarS1 ELISA for detecting antibodies against US PEDV S-INDEL mutant strains under the conditions of this study.

[0165] Antibodies against the US prototype strain and antibodies against the US S-INDEL mutant strain neutralized both virus strains to similar titers. Currently, the VN test based on the US PEDV prototype strain being performed in clinics can be used to detect antibodies against both US PEDV strains.

[0166] Pigs inoculated with both the prototype and S-INDEL mutant PEDV developed detectable IFA and ELISA antibodies in serum starting from 14 DPI in this study. Symmetrically, pigs in both groups developed low levels of serum neutralizing antibodies starting from 7 DPI. The IFA and ELISA assays in this study detected IgG antibodies: the VN test was able to potentially detect any antibody isotype with neutralizing activity. Whether this contributed to the early detection of the low levels of VN antibodies observed is unclear. In previous studies, PEDV VN antibodies have been reported to be detectable as early as 7 DPI

[20] .

[0167] The obvious genetic differences between the US prototype PEDV and the S-INDEL mutant PEDV are located in the S1 region (nucleotides 1 to 2214 corresponding to aa1 to 738, which match the positions in the prototype strain US / IN19338 / 2013, GenBank accession number KF650371), especially the N-terminal region of the S gene (nucleotides 1 to 1170 corresponding to aa1 to 390), while the rest of the genome is relatively conserved between the two US strains [4, 8, 10]. The PEDV prototype strain S1 protein used in the ProS1 ELISA and the PEDV S-INDEL mutant strain S1 used in the VarS1 ELISA had 92% amino acid identity. The reported PEDV neutralizing epitopes are located at amino acid residues 499 to 638, 744 to 759, 756 to 771, and 1368 to 1374 of the S protein [1, 21]. The protein sequences at these positions with neutralizing epitopes inside are conserved between the US prototype PEDV and the S-INDEL mutant PEDV. This explains why antibodies against the two PEDV strains It can be explained whether the swine stocks have been cross-neutralized. The ProS1 and VarS1 ELISAs were developed using the recombinant PEDV S1 protein (aa1-738). Although the US prototype and S-INDEL mutant PEDVs have significant differences in aa1-390, the two stocks still have some common epitopes within this region. Furthermore, the recombinant S1 proteins of the two PEDV stocks have relatively conserved sequences in aa390-738 (including neutralizing epitopes within this region). These may be the reasons why the ProS1 and VarS1 ELISAs can detect antibodies against both US PEDV stocks, despite the possibility of different sensitivities between assays. The IFA antibody assay and ProWV ELISA should detect antibodies against multiple antigenic proteins of PEDV, and thus they are expected to detect antibodies against both the US prototype and S-INDEL mutant PEDVs. Considering that the nucleocapsid protein is rather conserved among PEDVs, an ELISA based on the nucleocapsid protein is expected to detect antibodies against both US PEDV stocks. The applicant also included one porcine antiserum against the classical European PEDV CV777 strain for evaluation, and the PEDV CV777 antibody was detected by all the serological assays evaluated in this study. However, the antisera against TGEV Purdue, PHEV, PDCoV, and PRCV did not show cross-reactivity in the PEDV serological assays evaluated in this study.

[0168] Lin et al.

[22] prepared hyperimmune porcine antisera against US PEDV prototype strains, US PEDV S-INDEL mutant strains, TGEV Purdue strain, and TGEV Miller strain, and examined them by cell culture immunofluorescence (CCIF) assay (similar to our IFA antibody assay) and fluorescence focus reduction virus neutralization (FFRVN) assay (similar to our VN test). Antisera against US PEDV prototype strains, S-INDEL mutant strains, and the European CV777 strain were all found to have cross-reactivity in CCIF and FFRVN assays. Our findings were consistent with their results. In addition to the similar serological assays used by Lin et al., we also evaluated the serological reactivity of PEDV by three PEDV ELISAs. We also examined sequential serum samples (0 - 28 DPI) from pigs experimentally infected with two US PEDV strains, providing useful information on the kinetics of PEDV antibody production in weaned pigs. An interesting finding in the study by Lin et al. was that the hyperimmune antiserum against TGEV Miller strain, but not TGEV Purdue strain, cross-reacted with all PEDV strains in the CCIF assay, but not in the FFRVN assay. Conclusion The data from this study suggest that antibodies against the US PEDV prototype strain and S-INDEL mutant strains cross-react and cross-neutralize both strains in vitro. Current serological assays based on the US PEDV prototype strain can detect antibodies against both US PEDV strains. However, the cross-protective effects of these two PEDV strains need to be determined by in vivo pig studies. Goede et al.

[23] showed that female pigs exposed to S-INDEL mutant PEDV infection 7 months earlier could provide partial protection to neonatal pigs challenged with the US PEDV prototype strain. However, more in vivo studies are needed to clarify whether the US PEDV prototype strain or the S-INDEL mutant strain or both should be used to develop a vaccine that provides protection against both PEDV strains circulating in US pigs (see Examples 5 and 6 for further studies on this point). References 1.Song D,Park B.2012.Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology,diagnosis,and va ccines.Virus Genes 44:167-175. 2.Stevenson GW,Hoang H,Schwartz KJ,Burrough ER,Sun D,Madson D,Cooper VL,Pillatzki A,Gauger P,Schmitt BJ,Koster LG,Killian ML,Yoon KJ.2013.Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs,lesions,and viral genomic sequences.J Vet Diagn Invest 25:649-654. 3. Pasick J, Berhane Y, Ojkic D, Maxie G, Embury-Hyatt C, Swekla K, Handel K, Fairles J, Alexandersen S. 2014. Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in Canada. Transbound Emerg Dis 61:397-410. 4. Vlasova AN, Marthaler D, Wang Q, Culhane MR, Rossow KD, Rovira A, Collins J, Saif LJ. 2014. Distinct Characteristics and Complex Evolution of PEDV Strains, North America, May 2013 - February 2014. Emerg Infect Dis 20. 5. International Committee on Taxonomy of Viruses. 2012. Virus Taxonomy: 2012 Release. world wide web at ictvonline.org / virusTaxonomy.asp?version=2012. 6. Huang YW, Dickerman AW, Pineyro P, Li L, Fang L, Kiehne R, Opriessnig T, Meng XJ. 2013. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. MBio 4: e00737 - 00713. 7. Chen Q, Li G, Stasko J, Thomas JT, Stensland WR, Pillatzki AE, Gauger PC, Schwartz KJ, Madson D, Yoon KJ, Stevenson GW, Burrough ER, Harmon KM, Main RG, Zhang J. 2014. Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States. J Clin Microbiol 52:234-243. 8. Wang L, Byrum B, Zhang Y. 2014. New variant of porcine epidemic diarrhea virus, United States, 2014. Emerg Infect Dis 20:917-919. 9. Zhang J, Chen Q, Gauger PC, Harmon KM, Yoon KJ. 2014. Reply to “classification of emergent U.S. strains of porcine epidemic diarrhea virus by phylogenetic analysis of nucleocapsid and ORF3 genes”. J Clin Mi crobiol 52:3511-3514. 10. Oka T, Saif LJ, Marthaler D, Esseili MA, Meulia T, Lin CM, Vlasova AN, Jung K, Zhang Y, Wang Q. 2014. Cell culture isolation and sequence analysis of genetically diverse US porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene.Vet Microbiol 173:258-269. 11.Marthaler D,Bruner L,Collins J,Rossow K.2014.Third strain of porcine epidemic diarrhea virus,United States.Emerg Infect Dis 20:2162-2163. 12.Hoang H,Killian ML,Madson DM,Arruda PH,Sun D,Schwartz KJ,Yoon KJ.2013.Full-Length Genome Sequence of a Plaque-Cloned Virulent Porcine Epidemic Diarrhea Virus Isolate (USA / Iowa / 18984 / 2013)from a Midwestern U.S.Swine Herd.Genome Announc 1.13.Lawrence PK,Bumgardner E,Bey RF,Stine D,Bumgarner RE.2014.Genome sequences of porcine epidemic diarrhea virus: in vivo and in vitro phenotypes.Genome Announc 2. 14. Gerber PF, Gong Q, Huang YW, Wang C, Holtkamp D, Opriessnig T. 2014. Detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect ELISA. Vet J 202:33 - 36. 15. Gimenez - Lirola LG. 2014. Porcine epidemic diarrhea virus antibody tests available at ISU VDL, Swine Health Monitoring Projecct, 9 / 26 / 2014 ed. 16. Gimenez - Lirola LG, Baum D, Bower L, Chen Q, Sun D, Johnson J, Kalkwarf E, Madson D, Magtoto R, Yoon KJ, Zhang J, Zimmerman J, Main R. 2014. Porcine epidemic diarrhea virus: antibody - based test development., p.34 - 37, The 22nd Annual Swine Disease Conference for Swine Practitioners, Ames, Iowa. Nov 13 - 14, 2014. 17. Madson DM, Magstadt DR, Arruda PH, Hoang H, Sun D, Bower LP, Bhandari M, Burrough ER, Gauger PC, Pillatzki AE, Stevenson GW, Wilberts BL, Brodie J, Harmon KM, Wang C, Main RG, Zhang J, Yoon KJ. 2014. Pathogenesis of porcine epidemic diarrhea virus isolate (US / Iowa / 18984 / 2013) in 3 - week - old weaned pigs.Vet Microbiol 174:60-68. 18.Okda F,Liu X,Singrey A,Clement T,Nels on J,Christopher-Hennings J,Nelson EA,Lawson S.2015.Development of an indirect ELISA,blocking ELISA,fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus.BMC Vet Res 11:180. 19.Fan JH,Zuo YZ,Shen XQ,Gu WY,Di JM.2015.Development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein.J Virol Methods. 20.Thomas JT,Chen Q,Gauger PC,Gimenez-Lirola LG,Sinha A,Harmon KM,Madson DM,Burrough ER,Magstadt DR,Salzbrenner HM,Welch MW,Yoon KJ,Zimmerman JJ,Zhang J.2015.Effect of porcine epidemic diarrhea virus infectious doses on infection outcomes in naive neonatal and weaned pigs.PLoS One (submitted). 21. Sun D, Feng L, Shi H, Chen J, Cui X, Chen H, Liu S, Tong Y, Wang Y, Tong G. 2008. Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein. Vet Microbiol 131:73 - 81. 22. Lin CM, Gao X, Oka T, Vlasova AN, Esseili MA, Wang Q, Saif LJ. 2015. Antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains. J Virol 89:3332 - 3342. 23. Goede D, Murtaugh MP, Nerem J, Yeske P, Rossow K, Morrison R. 2015. Previous infection of sows with a “mild” strain of porcine epidemic diarrhea virus confers protection against infection with a “severe” strain. Vet Microbiol 176:161 - 164. Example 4 In response to the current PEDV pandemic and the lack of an effective vaccine, the present invention now provides a great achievement of providing a modified live vaccine based on INDEL mutants (a further example of which is OH851, first described by the Ohio Department of Agriculture, L. Wang et al., Emerg. Infect. Dis., 2014, v. 20, pp. 917-919) that are effective against the spectrum currently prevalent worldwide and are cross-protective against so-called prototype viruses that have caused devastating losses to pig herds, such as the well-known US / Colorado / 2013 (whose sequence is available under GenBank accession number KF272920). The vaccine of the present invention, a completely novel and groundbreaking pharmaceutical, also provides considerable flexibility with respect to dosing and the timing of dosing, for example, administration to female pigs both before and during pregnancy, as well as to piglets born to naive female pigs It is effective for administration to piglets including suckling piglets. Efficacy regarding breeding sows is also provided. Consequently, such vaccines provide protection against challenge by PEDV, including prototype and newly emerging INDEL type isolates, in both adjuvanted and non-adjuvanted forms, to parity sows, gilts, piglets, adult pigs, and breeding sows. The vaccines of the present invention thus provide a protective effect that was previously impossible to achieve by successfully achieving the following. (1) Vaccination of parity sows and gilts during pregnancy for protection, prevention, assistance in prevention, or assistance in suppression against diseases caused by PEDV (anorexia, weight loss, dehydration, fever, diarrhea, vomiting, reduced lactation ability, reduced reproductive ability), (2) Vaccination of parity sows and gilts during pregnancy for protection, prevention, assistance in prevention, or assistance in suppression against diseases and death caused by PEDV in piglets, (3) Vaccination of pigs 1 day old or older for protection, prevention, assistance in prevention, or assistance in suppression against diseases caused by PEDV, (4) Booster vaccination administered to female pigs before subsequent farrowing or once a year, (5) Healthy pigs can be inoculated with PEDV MLV and then boosted with PEDV inactivated vaccine, and (6) Generally, protecting pigs from weight loss or failure to gain weight caused by PEDV, etc. Attenuation of the prototype strain USA / IN19338 / 2013 Since its emergence in the United States in April 2013, porcine epidemic diarrhea virus (PEDV) has rapidly spread across the country, causing an estimated 8 million pigs to die in the first year and resulting in economic losses of $900 million to $1.8 billion [1]. In addition, PEDV has recently emerged or re-emerged in many countries, including China, Japan, South Korea, the Philippines, Thailand, Vietnam, Canada, Mexico, Germany, Belgium, France, and Portugal [2-10]. Therefore, PEDV still remains a major challenge to the global swine industry. Currently, there are two commercially available PEDV vaccines (inactivated vaccine and RNA particle vaccine) in the United States. However, several experiments have shown that these PEDV vaccines induced good IgG and IgA immune responses in groups previously exposed to PEDV, but did not induce good IgA responses in naive pigs after vaccination [11-12]. Modified live virus (MLV) PEDV vaccines are more effective than inactivated vaccines or subunit vaccines for inducing mucosal immunity. However, such safe and effective MLV PEDV vaccines against newly emerging US strains do not currently exist. The purpose of this study was to characterize the genomic and pathogenic changes of a US virulent PEDV prototype isolate after serial passage in cell culture and to determine whether any of the resulting viruses could be potential MLV vaccine candidates against PED. Materials and Methods A cell culture isolate of the US PEDV prototype strain, US / IN19338 / 2013

[13] , previously isolated in our laboratory, was serially passaged 100 times in Vero cells. The pathogenic changes of the selected virus were evaluated in a neonatal pig model. Sixty piglets (5 days old) without PED virus and antibodies were randomly divided into six groups of 10 pigs each. The pigs were fed with substitute milk. Groups 1 to 5 (G1 to G5) were inoculated with PEDV US / IN19338 / 2013 (10 4Pigs in six groups (G1-G6) were orally inoculated with 10 mL of a virus suspension containing 100 TCID50 / mL, and pigs in six other groups (G7-G12) were administered the same volume of virus-free culture medium as a negative control (Table 6). Clinical observations were recorded. Rectal swabs were collected upon arrival and daily and examined by quantitative real-time RT-PCR based on the PEDV nucleocapsid gene. Five pigs from each group were necropsied at 3 and 7 days post-inoculation (DPI). Gross and microscopic lesions in the small intestine, cecum, and colon were examined, and immunohistochemical (IHC) staining was performed. The severity of microscopic lesions was classified as follows: 0 = no lesions, 1 = minimal villous atrophy, 2 = mild villous atrophy, 3 = moderate villous atrophy, and 4 = severe villous atrophy. IHC staining was scored as follows: 0 = no signal, 1 = minimal staining, 2 = mild staining, 3 = moderate staining, and 4 = intense staining. The full genomic sequences of the virus in the original tissue homogenate and at passages P3, P7, P9, P25, P50, P65, P75, and P100 were determined using next-generation sequencing technology

[13] . were scored as follows: 0 = no signal, 1 = minimal staining, 2 = mild staining, 3 = moderate staining, and 4 = intense staining. The full genomic sequences of the virus in the original tissue homogenate and at passages P3, P7, P9, P25, P50, P65, P75, and P100 were determined using next-generation sequencing technology

[13] .

[0169]

Table 6

[0170] Results and Discussion Upon serial passage, the virus became adapted to cell culture. Higher passages of PEDV USA / IN19338 / 2013 at P50, P75, and P100 grew more efficiently and achieved higher infectious titers (Figure 17).

[0171] Negative control piglets remained negative throughout the test period. All piglets inoculated with P7, P25, P50, P75, or P100 virus presented with watery diarrhea at 1 - 4 DPI and mild to moderate diarrhea at 5 - 7 DPI. All piglets inoculated with P7, P25, P50, P75, or P100 virus excreted the virus in rectal swabs, and piglets inoculated with higher passages tended to excrete less virus (Figure 18). Specifically, the virus excretion levels were P7 > P25 > P50, P75, P100 > negative control (> means significantly higher, but there was no significant difference among the P50, P75, and P100 groups). Piglets inoculated with lower passages tended to have more severe villous atrophy. As shown in Figure 19, at 3 DPI, the severity of microscopic lesions in the duodenum was P7, P25 > P50, P75, P100, negative control; in the jejunum was P7, P25, P50 > P75, P100 > negative control; and in the ileum was P7, P25, P50 > P75, P100, negative control. The villus height to crypt depth ratio at 3 DPI in the jejunum was P7, P25, P50 < P75, P100 < negative control, and in the ileum was P7, P25, P50 < P75, P100, negative control (Figure 20). At 3 DPI, IHC staining in the jejunum was P7, P25, P50, P75 > P100 > negative control, and in the ileum was P7, P25, P50 > P75, P100, negative control, but there was no significant difference in IHC scores among the various viruses in the cecum or colon (Figure 21). The differences between groups at 7 DPI were not obvious.

[0172] The whole - genome sequences were not only for passages P7, P25, P50, P75, P100, but also for the original set We also determined for the woven homogenate and the viruses of passages P3, P9, and P65. As shown in Figure 16, by comparing the full-genome sequences, the nucleotide and putative amino acid changes during continuous passages up to P100 were mainly located in the replicase non-structural proteins (nsp) 2, nsp3, nsp4, nsp5, nsp6, nsp15, spike (S), ORF3, envelope (E), membrane (M), and nucleocapsid (N) proteins. Starting from P25, a point mutation of 24908A>T occurred in ORF3, and it is noteworthy that this resulted in a truncated ORF3 protein due to a premature termination codon in the putative amino acid translation: this point mutation was carried over to the virus of P100. Considering both the sequence changes during continuous passages and the changes in virus virulence, the amino acid changes at 15 positions may potentially be associated with virus attenuation during continuous passages in cell culture. These 15 positions include pp1a protein 1564Ser>Phe, 1896Thr>Ile, 2600Asn>Tyr, 3247Leu>Phe, and 3473Ala>Val; S protein 326Thr>Ile, 491Asn>Tyr, 888Gly>Arg, 1277Leu>Phe, 1399Ile>Thr, and 1358Cys>Leu; truncated ORF3 translation; E protein 69Leu>Ile; M protein 208Ala>Thr; and N protein 439Thr>Ile.

[0173] In summary, the US / IN19338 / 2013, a virulent US PEDV prototype isolate, clearly showed reduced pathogenicity during serial passages in cell culture. Clinical observations, virus shedding in rectal swabs, histopathological lesions, and IHC staining data suggest that the P75 and P100 viruses were further attenuated compared to the P7, P25, and P50 viruses. However, since the P75 and P100 viruses can still cause diarrhea, they may not be sufficiently attenuated to be MLV vaccine candidates. Continuous passage of the virus in cell culture is necessary to obtain a sufficiently attenuated vaccine candidate virus. Sixteen amino acid changes are thought to be associated with virus attenuation under the conditions of this study. This study provides a strong basis for developing an MLV PEDV vaccine and for understanding the molecular mechanisms of virus attenuation.

[0174] One of ordinary skill in the art will immediately recognize that, despite the nucleotide and amino acid sequence changes and the possible occurrence of deletions and insertions between different isolates, any amino acid position in a PEDV protein can be mapped to the appropriate amino acid position in other isolates because sequence alignment techniques are well known. Preferred algorithms and alignment programs for this purpose include ClustalX version 2.0 (Larkin et al, Bioinformatics 23, 2947-2948 (2007)), BioEdit version 7.0.4.1 (Hall, Nucl Acids Symp Ser 41, 95-98 (1999)), and the Lasergene software suite (DNASTAR Inc, Madison WI), which includes the Clustal W algorithm for multiple sequence alignment to align sequences and compare sequence similarities and differences.

[0175] Figure 16 shows that the following amino acid changes are preferred amino acid changes for generating a properly attenuated PEDV isolate that exhibits significant safety while providing vaccine efficacy: from ORF1a and b, amino acid positions 814 (Val), 1076 (Val), 1564 (Phe), 1896 (Ilu), 2310 (His), 2600 (Tyr), 3247 (Phe), 3473 (Val), 3522 (Arg); from the spike gene, amino acid positions 257 (Asn), 326 (Ile), 375 (Phe), 491 (Tyr), 881 (Arg), 888 (Arg), 1277 (Phe), 1339 (Thr), 1358 (Leu); from ORF3, any nucleotide change that provides a stop codon corresponding to amino acid position 39 or later; from genomic region E, 69 (Ile) of the encoded envelope - protein; 208 (Thr) of the encoded membrane protein for genomic region M; and 141 (Leu), 418 (Glu), 424 (Asp), and 439 (Ile) of the encoded nucleocapsid protein for genomic region N. In addition to these changes, which can be used alone or in combination, one of ordinary skill in the art will recognize that equivalent amino acid changes are commonly available, for example, a positively charged residue can be substituted with a positively charged residue (Arg, Lys, His), a negatively charged residue can be substituted with a negatively charged residue (Glu, Asp), or equivalent substitutions can be made based on polarity or functional groups (e.g., Thr to Ser and vice versa; Try to Trp and vice versa; Ileu, Val, Leu, etc.). References 1. Paarlberg, P.L. 2014. Updated estimated economic welfare impacts of porcine epidemic diarrhea virus (PEDV). http: / / ageconsearch.umn.edu / bitstream / 174517 / 2 / 14-4.Updated%20Estimated%20Economic%20Welfare%20Impacts%20of%20PEDV.pdf. 2. Grasland, B., Bigault, L., Bernard, C., Quenault, H., Toulouse, O., Fablet, C., Rose, N., Touzain, F. and Blanchard, Y., 2015. Complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france in december 2014. Genome Announc 3. 3. Mesquita, J.R., Hakze-van der Honing, R., Almeida, A., Lourenco, M., van der Poel, W.H. and Nascimento, M.S., 2015. Outbreak of Porcine Epidemic Diarrhea Virus in Portugal, 2015. Transbound Emerg Dis 62, 586 - 8 4. Pasick, J., Berhane, Y., Ojkic, D., Maxie, G., Embury-Hyatt, C., Swekla, K., Handel, K., Fairles, J. and Alexandersen, S., 2014. Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in Canada. Transbound Emerg Dis 61, 397 - 410. 5. Puranaveja, S., Poolperm, P., Lertwatcharasarakul, P., Kesdaengsakonwut, S., Boonsoongnern, A., Urairong, K., Kitikoon, P., Choojai, P., Kedkovid, R., Teankum, K. and Thanawongnuwech, R., 2009. Chinese-like strain of porcine epidemic diarrhea virus, Thailand. Emerg Infect Dis 15, 1112 - 5. 6. Song, D. and Park, B., 2012. Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus Genes 44, 167 - 75. 7. Vui, D.T., Thanh, T.L., Tung, N., Srijangwad, A., Tripipat, T., Chuanasa, T. and Nilubol, D., 2015. Complete genome characterization of porcine epidemic diarrhea virus in Vi etnam. Arch Virol 160, 1931 - 8. 8. Stadler, J., Zoels, S., Fux, R., Hanke, D., Pohlmann, A., Blome, S., Weissenbock, H., Weissenbacher - Lang, C., Ritzmann, M. and Ladinig, A., 2015. Emergence of porcine epidemic diarrhea virus in southern Germany. BMC Vet Res 11, 142. 9. Theuns, S., Conceicao-Neto, N., Christiaens, I., Zeller, M., Desmarets, L. M., Roukaerts, I. D., Acar, D. D., Heylen, E., Matthijnssens, J. and Nauwynck, H. J., 2015. Complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium, january 2015. Genome Announc 3. 10. Vlasova, A. N., Marthaler, D., Wang, Q., Culhane, M. R., Rossow, K. D., Rovira, A., Collins, J. and Saif, L. J., 2014. Distinct Characteristics and Complex Evolution of PEDV Strains, North America, May 2013 - February 2014. Emerg Infect Dis 20, 1620 - 1628. 11. Thomas, P. (2014). Field experiences using porcine epidemic diarrhea virus (PEDV) vaccine in herds experiencing endemic disease. In “2014 Iowa State University Swine Disease Conferencee for Swine Practitioners”, pp.38 - 42, Ames, Iowa. 12. Schwartz, T. J. and Rademacher, C. J. (2015). Evaluation of the effects of PEDv vaccine on PEDv naive and previously PEDv-exposed sows in a challenge model comparing immune response and preweaning mortality. In “2015 ISU James D. McKean Swine Disease Conferencee”, pp. 36-40, Ames, Iowa. 13. Chen, Q., Li, G., Stasko, J., Thomas, J. T., Stensland, W. R., Pillatzki, A. E., Gauger, P. C., Schwartz, K. J., Madson, D., Yoon, K. J., Stevenson, G. W., Burrough, E. R., Harmon, K. M., Main, R. G. and Zhang, J., 2014. Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States. J Clin Microbiol 52, 234-43. 14. Thomas, J. T., Chen, Q., Gauger, P. C., Gimenez-Lirola, L. G., Sinha, A., Harmon, K. M., Madson, D. M., Burrough, E. R., Magstadt, D. R., Salzbrenner, H. M., Welch, M. W., Yoon, K. J., Zimmerman, J. J. and Zhang, J., 2015. Effect of Porcine Epidemic Diarrhea Virus Infectious Doses on Infection Outcomes in Naive Conventional Neonatal and Weaned Pigs. PLoS O ne 10,e0139266. Attenuation of the U.S. S-INDEL mutant strain U.S. / IL20697 / 2014 (2014020697) Figure 24 and Tables 1 and 2 provide specific information regarding amino acid changes corresponding to the attenuated form of the mutant strain U.S. / IL20697 / 2014. The mutant isolate U.S. / IL20697 / 2014 was serially passaged in cell culture in two independent lineages.

[0176] In the first lineage, the virus was passaged in cell culture up to passage 60 (P60): the viruses of different passages were named 2014020697-P1, 2014020697-P2, 2014020697-P3…2014020697-P60. Among them, the full genomic sequences of viruses 2014020697-P3 (SEQ ID NO: 63), 2014020697-P5 (SEQ ID NO: 8), 2014020697-P7 (SEQ ID NO: 62), 2014020697-P18 (SEQ ID NO: 64), 2014020697-P30 (SEQ ID NO: 65), 2014020697-P45 (SEQ ID NO: 39), and 2014020697-P60 (SED ID NO: 66) were determined and compared.

[0177] In the second line, the virus was serially passaged in cell culture, and plaque (colony) purification was also performed on the virus of several passages. Among them, the whole genome sequences of virus 2014020697-P3R1 (SEQ ID NO: 67), 2014020697-P5R1 (SEQ ID NO: 68), 2014020697-P7R1 (SEQ ID NO: 15), 2014020697-P8R1 (SEQ ID NO: 35), 2014020697-P18R1 clone 89G8b (SEQ ID NO: 36), 2014020697-P18R1 clone 94F6a (SEQ ID NO: 37), and 2014020697-P18R1 clone 92F6a (SEQ ID NO: 37) (note that P18R1 94F6a and P18R1 92F6a (SEQ ID NO: 37) are replicates of the same clone executed for verification purposes: see Tables 1 and 2) were determined and compared. Furthermore, when 2014020697-P18R1 G8b and 2014020697-P18R1 F6a were passaged to the subsequent 19th and 20th passages, they maintained the genetic identity to their respective clones in passage 18R1. The resulting viruses both provided the required clinical safety by all recognized clinical trial endpoints, were highly protective against challenge by similar mutant (INDEL) strains, and remained cross-protective against highly pathological prototype strains.

[0178] Referring to the "Amino Acid Changes" section of FIG. 24 and Table 2, it can be seen that the ORF1a / 1b at amino acid position 551 encodes leucine. In fact, leucine is considered to be almost invariant in PEDV isolates at this position (whether prototype or mutant INDEL), in that leucine was found to represent residue 551 out of approximately 500 randomly selected compared to the published sequence. The clinical data for 2014020697-P18R1 F6a (SEQ ID NO: 37) and 2014020697-P18R1 G8b (SEQ ID NO: 36) both show significant levels of safety and efficacy, but it should be noted that 2014020697-P18R1 F6a is a material with high commercialization potential as it provides further enhancement in both safety and efficacy. 2014020697-P18R1 F6a provides proline at position 551, an amino acid known to disrupt or define the boundaries between domain types of peptide secondary structure, and thus Pro 551 is expected to contribute to the final phenotype. Given the invariant use of Leu at this position in the PEDV genome, it is a further embodiment of the invention to insert a Pro residue at position 551 or immediately adjacent thereto (within about 2 to 3 amino acid residues upstream or downstream thereof). Modeling using well-known algorithms (P.Y. Chou et al., "Prediction of Protein Conformation", Biochemistry, 13(2), pp222-245, 1974; J. Garnier et al., J. Mol. Biol., v120.pp.97-12 (See, e.g., 0,1978; and J. Garnier et al., Methods Enzymology, v266, pp. 540-543, 1996), when predicting the α-helix domain at the least involved residue DEDAT immediately upstream of position 551 of L, L is located at approximately the C-terminus of this secondary domain structure feature (however, still contributing to it). The insertion of proline substantially disrupts this α-helix feature. Similarly in this case, expecting to obtain a highly attenuated yet still safe vaccine, it is also within the practice of the present invention to insert a glycine residue as amino acid 551 of ORF1a / b, and the glycine residue can similarly be located within about 2 to 3 amino acid residues at position 551 upstream or downstream. All such substitutions are applicable to all PEDV clones, whether they are prototypes or mutants (INDELs).

[0179] Next, referring to the amino acid changes reflected in both 2014020697-P18R1 G8b and 2014020697-P18R1 F6a at position 973 of the spike protein, it is found that the wild-type amino acid tyrosine is substituted by histidine, and this substantially contributes to the useful phenotypes of these clones. Therefore, it is an embodiment of the present invention to generally provide histidine at this locus among all genomes of PEDV modified or selected to provide a safe and effective vaccine, whether derived from the prototype or from mutant (INDEL) strains.

[0180] Referring further to the important amino acid changes reflected in both 2014020697 - P18R1 G8b and 2014020697 - P18R1 F6a at position 1009 of the spike protein (i.e., immediately following NIT of SEQ ID NO: 47), it can be seen that the wild - type amino acid serine is replaced by proline. This mutation appeared during the initial attenuation passage between 2014020697 - P5R1 (SEQ ID NO: 68) and 2014020697 - P7R1 (SEQ ID NO: 15). Similar to the leucine - to - proline mutation described above in relation to position 551 of ORF1a / 1b, the appearance of proline greatly disrupts the secondary and tertiary protein structures and substantially contributes to the attenuated characteristics of the vaccine strain of the present invention. Similarly, in the context of the present invention, it is within the scope of the present invention to provide a proline residue at position 1009, or the corresponding position, in the spike protein of any PEDV vaccine virus. Glycine may also be substituted with proline at this position in any PEDV strain to improve the safety of the vaccine. Thus, in all genomes of PEDV modified or selected to provide a safe and effective vaccine, whether derived from a prototype or a mutant (INDEL) strain, it is an embodiment of the present invention to generally provide proline or glycine at this locus. It should also be noted that the proline / glycine substitution may also be made within about 2 - 3 amino acid residues immediately adjacent to position 1009, for example, upstream or downstream thereof.

[0181] Many additional features of the viruses of the invention useful as vaccines (including the passage 18R1 clones of G8b and F6a, SEQ ID NOs: 36 and 37, and another strain of virus represented by passage 38, SEQ ID NO: 78) are, among others, the expression of only the truncated ORF3 protein caused by a frameshift mutatio...

Claims

**Claim 1** A DNA polynucleotide that is at least 98% identical to SEQ ID NO: 67 at the full-length nucleotide level, and (a) P at position 1009 of the spike protein determined by reference to SEQ ID NO: 47 (immediately after NIT), and (b) H at position 973 of the spike protein determined by reference to SEQ ID NO: 47 (immediately after PFS), and (c) a modification of the coding sequence of ORF3 that results in premature termination of translation of ORF3, An isolated porcine epidemic diarrhea virus (PEDV) encoded by a DNA polynucleotide comprising the same. **Claim 2** The virus according to claim 1, wherein the DNA polynucleotide encoding the virus is at least 99% identical to SEQ ID NO: 67 at the full-length nucleotide level. **Claim 3** The virus according to claim 1, wherein the DNA polynucleotide encoding the virus is at least 99.5% identical to SEQ ID NO: 67 at the full-length nucleotide level. **Claim 4** A full-length RNA polynucleotide corresponding to the DNA polynucleotide encoding the isolated porcine epidemic diarrhea virus (PEDV) according to claim 1, or its complement. **Claim 5** The RNA polynucleotide according to claim 4, which is an infectious clone. **Claim 6** A plasmid or bacterial artificial chromosome comprising the DNA polynucleotide encoding the isolated porcine epidemic diarrhea virus (PEDV) according to claim 1. **Claim 7** Furthermore, (d) L or P at position 551 of the polyprotein 1a / 1b determined by reference to SEQ ID NO: 46 (immediately after DAT) The isolated porcine epidemic diarrhea virus (PEDV) according to claims 1-3, comprising the same. **Claim 8** The isolated porcine epidemic diarrhea virus (PEDV) according to claim 7, comprising a modification of the coding sequence of ORF3 that results in premature termination of translation of ORF3, wherein the modification of ORF3 comprises a deletion that generates the amino acids LTA NPL at positions 138-143 (immediately after NGKAA of SEQ ID NO: 48), and cleavage of the ORF protein after position 143. **Claim 9** A full-length RNA polynucleotide corresponding to the DNA polynucleotide encoding the isolated porcine epidemic diarrhea virus (PEDV) according to claim 7 or 8, or its complement. **Claim 10** The RNA polynucleotide according to claim 9, which is an infectious clone. **Claim 11** A plasmid or bacterial artificial chromosome comprising a DNA polynucleotide encoding the isolated porcine epidemic diarrhea virus (PEDV) according to claim 7.

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