Porcine reproductive and respiratory syndrome chimeric virus strain and vaccine composition using the same

Abstract

The present invention relates to a novel attenuated North American PRRS chimeric virus produced by substituting some genes and deglycosylating specific genes of North American PRRS Lineage 1, which is prevalent in Korea, and to the use thereof. The attenuated North American PRRS chimeric virus of the present invention has reduced proliferative capacity in pigs compared to the existing wild-type virus, but forms cellular and humoral immunity at the same level as the field virus, so it can be utilized as a vaccine with guaranteed stability.

Description

Technology Field

[0001] The present invention relates to an attenuated chimeric virus of Lineage 1 of North American porcine reproductive and respiratory syndrome virus (PRRSV-NA), a vaccine composition for the prevention and treatment of North American porcine reproductive and respiratory syndrome using the same, and a method for manufacturing the same. Background Technology

[0003] Porcine reproductive and respiratory syndrome virus (PRRSV) is a virus classified in the order Nidovirales, family Arteriviridae, and genus Arterivirus, and is a pathogen that causes porcine reproductive and respiratory syndrome.

[0004] The above viral disease was first discovered in the United States in 1987 (North American type PRRS), followed by discovery in Europe (European type PRRS), and was identified in Asia in the early 1990s. To date, the porcine genital and respiratory syndrome virus has spread globally with endemic characteristics in pig-producing countries, causing massive economic losses every year.

[0005] PRRSV is classified into two genotypes (Type 1 / European and Type 2 / North American) based on genetic differences, and there is approximately 50–70% nucleotide sequence homology and 50–80% amino acid homology between these two genotypes. Within each genotype, it is known that there are genetic differences in nucleotide sequences ranging from an average of 12.5–15% to a maximum of 21–30%. The PRRSV gene is a single-strand, positive-sense RNA with a size of approximately 15 Kb and consists of nine overlapping open reading frames (ORFs: 1a, 1b, 2a, 3, 4, 5, 6, and 7). Additionally, a 5'UTR (non-structural gene) is located before ORF1, and a 3'UTR (non-structural gene) is located after ORF7. ORF1a and ORF1b code for 14 non-structural proteins required for viral replication and transcription. Encapsulated viral particles contain three important structural proteins: the glycosylated envelope protein (GP5, 25 kDa), the unglycosylated membrane protein (M, 18 kDa), and the nucleocapsid protein (N, 15 kDa); these structural proteins are encoded by ORF5, ORF6, and ORF7, respectively. The GP5 protein is known to be involved in the recognition of host cell receptors and, along with the M protein, participates in the formation of PRRSV neutralizing antibodies. The M protein has been reported to be the most genetically conserved among the structural proteins of PRRSV and is known to play an important role in viral assembly and budding.

[0006] Porcine Reproductive and Respiratory Syndrome (PRRS) is the infectious disease causing the greatest damage to the domestic pig farming industry. PRRS causes reproductive disorders in pregnant sows, including infertility, miscarriage, premature birth, and stillbirth, while inducing respiratory symptoms such as sneezing and fever in suckling pigs and fattening pigs. Generally, severe respiratory symptoms are caused by secondary infections, such as bacteria, following viral infection; however, in cases of chronic infection, reduced weight gain and increased mortality rates occur without characteristic clinical symptoms. The proliferation of PRRSV varies depending on the age of the infected pig. In weaned piglets, the virus is generally cleared from the bloodstream around 6 weeks after infection, although there have been cases where it was detected in lymph nodes or tonsils for up to 156 days. In pregnant sows and boars, the virus disappears from the bloodstream within 4 weeks of infection; however, due to infection in the reproductive system, it can be intermittently detected in boar semen for 43 to 92 days after infection.

[0007] It is known that infection with the North American PRRS virus causes higher pathogenicity than infection with the European PRRS virus. However, when classified by genetic characteristics, pathogenicity and transmissibility are determined by each genotype subgroup rather than by distinguishing between North American or European types.

[0008] According to the domestic PRRS virus variant and pathogenicity monitoring report (2016), among the various PRRS virus strains causing infection in Korea, PRRSV-Lineage 5 was dominant from 2014 (46.7%) to 2019 (31.1%), while Lineage 1 strains were the second most common strain from 2014 (1.8%) to 2019 (29.6%). The Lineage 1 group includes virulent strains such as NADC30 and MN184, and many field isolates of Lineage 1 have also been reported to be virulent. In the United States, Prevacent PRRS (Elanco, Indianapolis, IN, USA), a PRRSV MLV vaccine derived from the Lineage 1 variant, has been commercially available since 2018, but there is currently no Lineage 1 vaccine available in Korea. Therefore, a new vaccine against the North American type PRRS Lineage 1 virus is required in Korea to control this disease.

[0009] Due to the importance of this disease, despite significant efforts and investments over the past 20 years since the discovery of the porcine genital-respiratory syndrome (RPRS) virus—the causative agent of the disease—an effective vaccine has yet to be developed. Although various vaccines, including inactivated and live attenuated vaccines, have been developed to prevent RPRS, it has been found that only attenuated vaccines with guaranteed infectivity induce a satisfactory level of protective effect.

[0010] However, because porcine genital and respiratory syndrome viruses exist with great genetic diversity and cross-immunity between the various viruses is not well formed, it is difficult to defend against various types of porcine genital and respiratory syndrome viruses with a single vaccine. Furthermore, since attenuated vaccines can currently only be produced through more than 100 to 200 consecutive passages in cell lines of other animal species, the development period is long. Also, viruses that are attenuated in this way and used as vaccines often multiply within the pig's body and regain pathogenicity, failing to prevent the disease in pigs vaccinated with the attenuated vaccine and instead causing the disease, making it difficult to guarantee safety. Prior art literature

[0012] U.S. Patent No. 7608272 (October 27, 2009) The problem to be solved

[0013] The present invention aims to provide a novel attenuated North American PRRS Lineage 1 chimeric virus and a method for producing the same through attenuation by partial gene substitution and / or specific gene deglycosylation.

[0014] The purpose is to provide a vaccine effective for the prevention and / or treatment of North American type porcine reproductive and respiratory syndrome based on the above-mentioned chimeric virus, and a method for manufacturing the same.

[0015] In addition, the present invention aims to provide a method for preventing and / or treating porcine reproductive and respiratory syndrome, comprising the step of administering the vaccine composition. means of solving the problem

[0017] The present invention provides a vector for producing porcine reproductive and respiratory syndrome virus (CNSA11-Lineage1).

[0018] The above vector may include one or more of the nucleotide sequences of SEQ ID NOs 9 to 14.

[0019] The present invention provides a vector for producing porcine reproductive and respiratory syndrome chimeric virus (CNSA11-Lineage1 chimeric virus) characterized by including the nucleotide sequence of SEQ ID NO. 1 and the nucleotide sequence of SEQ ID NO. 15 being changed to the nucleotide sequence of SEQ ID NO. 16.

[0020] The present invention provides a vector for producing porcine reproductive and respiratory syndrome chimeric virus (CNSA11-Lineage1-N44S chimeric virus) characterized by including the nucleotide sequence of SEQ ID NO. 1 and changing the nucleotide sequence of SEQ ID NO. 16 to the nucleotide sequence of SEQ ID NO. 17.

[0021] The present invention provides a novel polynucleotide for producing porcine reproductive and respiratory syndrome virus.

[0022] In one embodiment, the polynucleotide may be one or more selected from the nucleotide sequences of SEQ ID NOs. 9 to 14.

[0023] In one embodiment, the polynucleotide may be one or more selected from the nucleotide sequences of SEQ ID NOs. 15 and 16.

[0024] In one embodiment, the polynucleotide may be the base sequence of SEQ ID NO. 17.

[0025] The present invention provides a novel polypeptide for the porcine reproductive and respiratory syndrome chimeric virus.

[0026] In one embodiment, the polypeptide may be one or more selected from the amino acid sequences of SEQ ID NOs. 18 and 19.

[0027] The present invention provides a polypeptide for increasing the production rate of neutralizing antibodies against porcine reproductive and respiratory syndrome chimeric virus (CSNA11-L1-N44S chimeric virus).

[0028] In one embodiment, the polypeptide may be the amino acid sequence of SEQ ID NO. 19.

[0029] The present invention provides a method for producing porcine reproductive and respiratory syndrome virus, comprising the step of inserting any one of the above vectors into a cell.

[0030] The above cell may be MARC-145, but is not limited thereto.

[0031] The present invention provides a method for producing a porcine reproductive and respiratory syndrome chimeric virus (CSNA11-Lineage1 chimeric virus), characterized by including the step of substituting the nucleotide sequence of SEQ ID NO. 15 with the nucleotide sequence of SEQ ID NO. 16 in a vector for producing a porcine reproductive and respiratory syndrome virus (CSNA11).

[0032] The present invention provides a method for producing a porcine reproductive and respiratory syndrome chimeric virus (CSNA11-Lineage1-N44S chimeric virus), characterized by including the step of substituting Adenosine (A) at position 38 in the amino acid sequence of SEQ ID NO. 16 with Guanine (G) in a vector for producing a porcine reproductive and respiratory syndrome virus (CSNA11-Lineage1 chimeric virus).

[0033] The present invention provides a method for producing a porcine reproductive and respiratory syndrome chimeric virus (CSNA11-Lineage1-N44S chimeric virus), characterized by comprising: (a) a step of producing a vector containing the nucleotide sequence of SEQ ID NO. 1; (b) a step of substituting the nucleotide sequence of SEQ ID NO. 15 in the produced vector with the nucleotide sequence of SEQ ID NO. 16; and (c) a step of substituting Adenosine (A) at number 38 in SEQ ID NO. 16 with Guanine (G).

[0034] The present invention provides a porcine reproductive and respiratory syndrome virus produced by the above method.

[0035] The present invention provides a cell transformed with the above vector.

[0036] The present invention provides a vaccine composition for the prevention or treatment of porcine reproductive and respiratory syndrome comprising the above-mentioned chimeric virus as an active ingredient.

[0037] The above vaccine composition may additionally include an adjuvant. Effects of the invention

[0039] The present invention relates to a North American PRRS Lineage 1 chimeric virus attenuated through reverse genetics using a highly proliferative North American PRRS virus strain isolated in Korea. The attenuated North American PRRS Lineage 1 chimeric virus of the present invention has the effect of forming antibodies similar to the wild-type virus in the serum after inoculation with pigs, maintaining a low viral content in the blood before disappearing early, and not causing severe lesions in tissues.

[0040] Therefore, the novel attenuated PRRS chimeric virus of the present invention can be usefully utilized in the manufacture of live attenuated vaccines for the prevention and treatment of porcine reproductive and respiratory syndrome. Brief explanation of the drawing

[0042] Figure 1 is a photograph showing the results of IFA for chimeric virus replication in MARC-145 cells. Figure 2 is a photograph showing the results of IFA on chimeric virus replication in PAM cells. Figure 3 is a graph showing the results of body temperature measurements in the experimental group evaluating the safety and immunogenicity of the CSNA11-L1 chimeric virus. Figure 4 is a graph showing the results of body weight measurements of the experimental group evaluating the safety and immunogenicity of the CSNA11-L1 chimeric virus. Figure 5 is a graph showing the results of measuring blood viral content in the experimental group for the safety and immunogenicity evaluation of the CSNA11-L1 chimeric virus. Figure 6 is a graph showing the results of the cellular immunity evaluation of the experimental group for the safety and immunogenicity evaluation of the CSNA11-L1 chimeric virus. Figure 7 is a graph showing the results of antibody measurement in the experimental group for the safety and immunogenicity evaluation of the CSNA11-L1 chimeric virus. Figure 8 is a graph showing the results of body temperature measurements in the experimental group for the efficacy evaluation of the CSNA11-L1-N44S chimeric virus. Figure 9 is a graph showing the results of body weight measurements in the experimental group for the efficacy evaluation of the CSNA11-L1-N44S chimeric virus. Figure 10 is a graph showing the results of comparing blood viremia levels after challenge inoculation in the experimental group for the efficacy evaluation of the CSNA11-L1-N44S chimeric virus. Figure 11 is a graph showing the antibody measurement results of the experimental group for the efficacy evaluation of the CSNA11-L1-N44S chimeric virus. Figure 12 is a graph showing the results of neutralizing antibody measurements in the experimental group for the efficacy evaluation of the CSNA11-L1-N44S chimeric virus. Figure 13 is a graph showing the lung tissue lesion index of the experimental group for the efficacy evaluation of the CSNA11-L1-N44S chimeric virus. Specific details for implementing the invention

[0043] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be embodied in various forms and is not limited to the embodiments and examples described herein.

[0044] Throughout this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0045] The term "Porcine Respiratory and Reproductive Syndrome" used in the present invention is an infectious disease caused by PRRSV, which causes reproductive disorders such as infertility, miscarriage, premature birth, and stillbirth in pregnant sows, and causes respiratory symptoms such as sneezing and fever in suckling pigs and fattening pigs, and is also referred to as "PRRS".

[0046] The term "Porcine Respiratory and Reproductive Syndrome Virus" used in the present invention is a virus classified in the order Nidovirales, family Arteriviridae, genus Arterivirus, which is a pathogen causing Porcine Respiratory and Reproductive Syndrome, and is also referred to as "PRRS virus" or "PRRSV".

[0047] The term "chimeric virus" as used in the present invention refers to a virus having at least one sequence substituted, and is also referred to as a "chimeric virus."

[0048] The term "infectious clone" used in the present invention is a polynucleotide that can induce the production of a virus when inserted into a cell, and is also referred to as an "infectious clone."

[0049] The term "MARC-145" as used in this invention is a monkey kidney cell line with ATCC Accession No. CRL-12230.

[0050] The term "GP5 protein" as used in this invention is a polypeptide encoded by ORF5 of the PRRSV genome, which is involved in the recognition of receptors in host cells and, together with the M protein, is involved in the formation of PRRSV neutralizing antibodies.

[0051] The term "ectodomain" as used in this invention refers to a domain of a membrane protein located in the extracellular space.

[0052] The term "CNSA11" used in this invention refers to one of the strains of PRRSV, which has low pathogenicity and high proliferative capacity.

[0053] Sequence No. 1 of the present invention is the full-length gene of CSNA11.

[0054] The term "Lineage 1" used in the present invention refers to a group of classifications of PRRSV, and the Lineage 1 group includes toxic strains such as NADC30 and MN184.

[0055] The term "CSNA11-Lineage1 chimeric virus" used in the present invention is a chimeric virus in which the major neutralizing epitope is replaced with the sequence of Lineage 1 by replacing the ORF5 ectodomain sequence (Sequence No. 15) of the CSNA11 infectious clone with the ectodomain sequence (Sequence No. 16) of CSNA11-Lineage1.

[0056] The term "CSNA11-Lineage1-N44S chimeric virus" used in the present invention is a chimeric virus in which the nucleotide at position 38 of the ORF5 ectodomain of the CSNA11-L1 virus is changed to a nucleotide sequence (Sequence No. 17) in which Adenosine (A) is substituted with Guanine (G), and due to the change in nucleotide sequence, the amino acid sequence (Sequence No. 19) in which the amino acid at position 44 of Asparagine (N) is substituted with Serine (S) in the ORF5 amino acid sequence (Sequence No. 18) of the CSNA11-L1 virus has an amino acid sequence (Sequence No. 19).

[0057] The term “attenuation” as used in this invention refers to a strain that has been modified to reduce its pathogenicity.

[0058] The term “vector” may mean a construct capable of delivering, preferably expressing, one or more gene(s) or sequence(s) of interest within a host cell. Examples include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated within liposomes, and certain eukaryotic cells, e.g., producer cells.

[0059] The term "immune response" as used in this invention means humoral immune response, cellular immune response, or all of these.

[0060] Inserting the gene of the above-mentioned PRRS virus into a vector can be performed by a method known in the art. Additionally, inserting the above-mentioned vector into a cell can be performed by a method known in the art.

[0061] As used in this invention, the term "prevention" refers to any act of suppressing or delaying the onset of a disease by administering the composition, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of an individual suspected of or suffering from a disease by administering the composition.

[0062] The present invention is to be explained in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0064] [Example 1]

[0065] Production of the complete gene sequence and infectious clone of the CSNA11 PRRS virus

[0067] The inventors produced an infectious clone of a stable CSNA11 PRRS virus backbone by the following method to produce a PRRS chimeric virus. Genetic information of the highly proliferative CSNA11 virus was obtained, and an infectious clone of the CSNA11 virus was produced based on this information.

[0068] The entire nucleotide sequence of the CSNA11 virus (15kb) was analyzed, and the nucleotide sequences of Sequence Nos. 2 to 8 were synthesized (GenScript) to produce the full length of the entire CSNA11 virus nucleotide sequence (15kb, Sequence No. 1). Among these, Sequence Nos. 2, 3, and 6 were synthesized by substituting only one nucleotide each so as not to affect the amino acid sequence of CSNA11, as restriction enzyme sequences were present. Additionally, at the end of each segment, a restriction enzyme site was designed to be present for infusion cloning to produce the full length gene according to the CSNA11 virus nucleotide sequence.

[0069] After TA cloning (Sequence No. 2) the nucleotide sequences of SEQ ID NOs. 2 to 8 into the pOptiVEC plasmid (Invitrogen) for virus production, SEQ ID NOs. 3 to 8 were sequentially inserted using an Infusion PCR kit (Takara, Japan) to obtain an infectious clone of 15kb. Subsequently, it was confirmed that the obtained infectious clone matched the nucleotide sequence of the original virus (rescued virus).

[0070] Sequence number nucleotide sequence 1 GCGCGC GGCGCC CGTACG 2 GCGCGT CCGCCACGCGT 3 GGCGCT TGCTCTTTTACCGATTTGCCGCCTTCAGATGGCGCGGATGCGGACGGGGGGGGGCCGTTTCGGACGGTAAAAAGAAAAGCTGAAAGGCTCTTTGACCAACTGAGCCGTCAGGTTTTTGACCTCGTCTCCCACCTCCCTGTTTTCTTCTCACGCCTTTTCAACCCTGGCGGTGGTTATTCTCCGGGTGATTGGGGTTTTGCAGCTTTTACTCTATTGTGCCTCTTTTTATGTTACAGTTACCCAGCCTTTGGTATTGCTCCCCTCTTGGGTGTATTTTCTGGGTCTTCTCGGCGTGTTCGAATGGGGGTTTTTGGCTGCTGGTTGGCTTTTGCTGTTGGTCTGTTCAAGCCTATGTCCGACCCAGTCGGCGCTGCTTGTGAGTTTGACTCGCCAGAGTGTAGAAACATCCTTCATTCTTTTGAGCTTCTCAAACCTTGGGACCCTGTTCGCAGCCTTGTTGTGGGCCCCGTCGGTCTCGGTCTTGCCATTCTTGGCAGGTTACTGGGCGGGGCACGCTATATCTGGCACTTTTTGCTTAGGCTTGGCATTGTTGCGGACTGTATCTTGGCTGGAGCTTACGTGCTTTCTCAAGGTAGGTGTAAAAAGTGCTGGGGATCTTGTATAAGAACTGCTCCTAATGAGGTTGCTTTTAACGTGTTTCCTTTCACACGCGCGACCAGGTCGTCACTTATCGACCTGTGCGATCGGTTTTGTGCGCCAAAAGGAATGGACCCCATTTTTCTCGCCACTGGGTGGCGCGGGTGCTGGGCCGGCC 4 GAAGCCCCATTGAGCAACCCTCTGAAAAACCCATCGCGTTTGCCCAATTGGATGAAAAGAAGATTACGGCTAGGACTGTGGTCGCCCAGCCTTATGACCCCAACCAAGCCGTAAAGTGCTTGCGGGTATTGCAGGCGGGTGGGGCGATGGTGGCTAAGGCGGTCCCAAAAGTGGTTAAGGTTTCCGCTGTCCCATTCCGAGCCCCCTTCTTTCCCACTGGAGTGAAAGTTGACCCTGATTGCAGGGTCGTGGTTGACCCTGACACTTTCACTGCAGCTCTCCGGTCTGGCTACTCCACCACAAACCTCGTCCTTGGTGTAGGGGACTTTGCCCAGTTGAATGGATTAAAAATCAGGCAAATTTCCAAGCCTTCAGGGGGAGGCCCACATCTCATGGCTGCCCTGCATGTTGCCTGCTCGATGGCTCTGCACATGCTTGCTGGGATTTATGTGACTGCGGTGGGTTCTTGCGGCACCGGCACCAACGACCCGTGGTGCGCCAACCCGTTTGCCGTTCCTGGCTACGGACCTGGCTCTCTCTGCACGTCTAGATTGTGCATTTCCCAACACGGCCTTACCCTGCCCTTGACAGCACTTGTGGCGGGATTCGGTATTCAAGAAATTGCCTTGGTCGTTTTGATTTTTGTTTCCATCGGAGGCATGGCTCACAGGTTGAGCTGTAAGGCTGACATGCTGTGTGTTTTGCTTGCAATTGCCAGCTATGTTTGGGTGCCTCTTACCTGGTTGCTTTGTGTGTTTCCTTGCTGGTTGCGCTGTTTTTCTTTGCACCCCCTCACCATCCTATGGTTGGTGTTTTTCTTGATTTCTGTGAATATGCCTTCAGGAATCTTGGCCATGGTGTTGTTGGTTTCTCTTTGGCTTCTTGGTCGTTATACTAATGTTGCTGGCCTTGTCACCCCCTACGACATTCATCATTACACCAGTGGCCCCCGCGGTGTTGCCGCCTTGGCTACCGCACCAGATGGGACCTACTTGGCCGCTGTCCGCCGCGCTGCGTTGACTGGCCGCACCATGCTGTTTACCCCGTCCCAGCTTGGGTCTCTTCTTGAGGGTGCTTTCAGAACTCGAAAGCCCTCACTGAACACCGTCAATGTGATCGGGTCCTCCATGGGCTCTGGCGGGGTGTTTACCATCGACGGGAAAGTCAAGTGTGTAACTGCCGCACATGTCCTTACGGGCAATTCAGCTCGGGTTTCCGGGGTCGGCTTCAATCAAATGCTTGACTTTGACGTAAAGGGAGATTTCGCTATAGCTGATTGCCCGAATTGGCAAGGGGCTGCCCCCAAGACCCAATTCTGCGCGGATGGATGGACTGGCCGTGCCTATTGGCTAACATCCTCTGGCGTCGAACCCGGCGTCATTGGAAAAGGATTCGCCTTCTGCTTCACCGCATGTGGCGATTCCGGGTCCCCAGTGATCACCGAGGCCGGTGAGCTTGTCGGCGTTCACACGGGATCGAATAAACAAGGGGGGGGCATTGTTACGCGCCCCTCAGGCCAGTTTTGTAATGTGGCACCCATCAAGCTAAGCGAATTAAGTGAATTCTTTGCTGGGCCTAAGGTCCCGCTCGGTGATGTGAAGGTCGGCAGCCACATAATCAAAGACATAGGCGAGGTGCCTTCAGATCTTTGTGCCTTGCTTGCTGCCAAACCTGAACTGGAAGGAGGCCTCTCCACCGTCCAACTTCTTTGTGTGTTTTTTCTCCTGTGGAGAATGATGGGACATGCCTGGACGCCCTTGGTTGCTGTGAGTTTCTTTATTTTGAATGAGGTTCTCCCTGCCGTCCTGGTCCGGAGTGTTTTCTCCTTTGGAATGTTTGTGCTATCCTGGCTCACGCCATGGTCTGCGCAAGTTCTGATGATCAGGCTTCTGACAGCTGCTCTTAACAGGAACAGATGGTCACTTGCCTTTTTCAGCCTCGGTGCAGTGACCGGTTTTGTCGCAGATCTTGCGGCCACTCAGGGGCATCCGTTGCAGGCAGTGATGAATTTGAGCACCTATGCATTCCTGCCTCGGATGATGGTTGTGACCTCACCAGTCCCAGTGATCACGTGTGGTGTCGTGCACCTACTTGCCATCATTTTGTACTTGTTTAAGTACCGTGGCCTGCACCATATCCTTGTTGGCGATGGAGTGTTCTCTGCGGCTTTCTTCTTGAGATACTTTGCCGAGGGAAAGTTGAGGGAAGGGGTGTCGCAATCCTGCGGAATGAATCATGAGTCTCTGACTGGTGCCCTCGCTATGAGACTCAATGACGAGGACTTGGATTTCCTTATGAAATGGACTGATTTTAAGTGCTTTGTTTCTGCGTCCAACATGAGGAATGCAGCGGGTCAATTTATCGAGGCTGCCTATGCTAAAGCACTTAGAGTGGAACTGGCCCAGTTGGTGCAGGTTGATAAAGTTCGAGGTACTTTGGCCAAACTTGAAGCTTTTGCTGATACCGTGGCACCTCAACTCTCGCCCGGTGACATTGTTGTCGCTCTCGGCCACACGCCTGTTGGCAGTATCTTCGACCTAAAGGTTGGTAGCACCAAGCATACCCTCCAAGCCATTGAGACCAGAGTCCTTGCTGGGTCCAAAATGACCGTGGCGCGC 5 GTCGTCGACCCGACCCCCACGCCCCCACCCGCACCCGTGCCCATCCCCCTCCCACCGAAGGTCCTGGAGAATGGCCCCAACGCTTGGGGGGATGAGGACCGTTTGAATAAGAAGAAGAGGCGCAGGATGGAAGCCCTCGGCATCTATGTTATGGGCGGGAAAAAGTACCAGAAATTTTGGGACAAGAATTCCGGTGATGTGTTTTATGAGGAGGTCCATAATAACACAGATGAGTGGGAGTGTCTTAGAGTTGGCGACCCTGCCGACTTTGACCCTGAGAAGGGAACTCTGTGTGGACATGTCACCATTGAAAACAAGGCTTACCATGTTTACACCTCCCCATCTGGTAAGAAGTTCTTGGTCCCCGTCAACCCAGAGAATGGAAGAGTTCAATGGGAAGCTGCAAAGCTTTCCGTGGAGCAGGCCCTAGGTATGATGAATGTCGACGGCGAACTGACTGCCAAAGAACTGGAGAAATTGAAAAGAATAATTGACAAACTCCAGGGCCTGACTAAGGAGCAGTGTTTAAACTGCTAGCCGCCAGCGACTTGACCCGCTGTGGTCGCGGCGGCTTGGTTGTTACTGAGACAGCGGTAAAAATAGTCAAATTTCACAACCGGACTTTCACCCTGGGACCTGTGAATTTAAAAGTGGCCAGTGAGGTTGAGCTAAAAGACGCGGTTGAGCACAACCAACACCCGGTTGCGAGACCAATCGAtGGTGGAGTTGTGCTCCTGCGTTCCGCGGTTCCTTCGCTTATAGACGTCTTGATCTCCGGTGCTGATGCATCTCCCAAGTTACTCGCCCATCACGGGCCGGGAAACACTGGGATCGAtGGCACGCTCTGGGATTTTGAGTCCGAAGCCACTAAAGAGGAAGTTGCACTCAGTGCGCAAATAATACAGGCTTGTGACATCAGGCGCGGCGACGCTCCTGAAATTGGTCTCCCTTACAAGCTGTACCCTGTTAGGGGTAACCCCGAGCGGGTGAAAGGAGTTCTGCAGAATACAAGGTTTGGAGACATACCTTACAAAACCCCCAGTGACACTGGAAGCCCAGTGCACGCGGCTGCCTGCCTTACGCCCAACGCCACTCCGGTGACAGATGGGCGCTCCGTCTTGGCCACGACCATGCCCCCCGGGTTTGAGTTATATGTACCGACCATACCAGCGACTGTCCTTGATTACCTTGACTCTAGGCCTGACTGCCCTAAACAGCTGACAGAGCACGGCTGCGAAGATGCCGCACTGAAAGACCTCTCTAAATATAACTTGTCCACCCAAGGCTTTGTTTTACCTGGAGTTCTTCGCCTTGTGCGGAAATACCTGTTTGCCCATGTAGGTAAGTGCCCACCCGTTCATCGGCCCTCTACTTACCCTGCTAAGAATTCTATGGCTGGAATAAATGGGAACAGGTTCCCAACCAAGGACATTCAGAGCGTCCCTGAAATCGACGTTCTGTGCGCACAGGCTGTGCGAGAAAACTGGCAAACTGTCACCCCTTGTACTCTTAAGAAACAGTACTGCGGGAAGAAGAAGACTAGGACCATACTCGGCACCAATAACTTCATCGCACTAGCCCACCGAGCAGTGTTGAGTGGTGTTACCCAGGGCTTCATGAAAAAGGCGTTTAACTCGCCCATCGCCCTCGGAAAGAACAAGTTTAAGGAGCTACAGACTCCGGTCCTGGGCAGGTGCCTTGAAGCTGATCTCGCATCCTGCGATCGATCCACGCCTGCAATTGTCCGCTGGTTTGCCGCCAACCTTCTTTATGAACTTGCCTGCGCTGAAGAGCATCTACCGTCGTACG 6 TGCTGAACTGCTGCCACGACCTACTGGTTACGCAGTCCGGCGCAGTGACTAAGAGAGGTGGCCTGTCGTCTGGCGACCCGATCACCTCTGTGTCTAACACCATTTATAGTTTGGTGATCTATGCACAGCATATGGTGCTTAGTTACTTCAAAAGTGGTCACCCCCATGGTCTTCTGTTCTTACAAGACCAGCTAAAGTTTGAGGACATGCTCAAGGTTCAACCCCTGATCGTCTATTCGGACGACCTCGTGCTGTATGCCGAGTCTCCCACCATGCCAAACTATCACTGGTGGGTTGAACATCTGAATTTGATGCTGGGGTTTCAGACGGACCCAAAGAAGACAGCAATAACAGACTCGCCATCATTTCTAGGCTGTAGAATAATAAATGGGCGGCAGCTAGTCCCCAACCGTGACAGGATCCTCGCGGCCCTCGCCTATCACATGAAGGCGAGTAATGTTTCTGAATACTATGCCTCAGCGGCTGCAATACTCATGGACAGCTGTGCTTGTTTGGAGTATGATCCTGAATGGTTTGAAGAACTTGTAGTTGGAATAGCGCAGTGCGCCCGCAAGGACGGCTACAGCTTTCCCGGCACGCCGTTCTTCATGTCCATGTGGGAAAAACTTAGGTCCAATTATGAGGGGAAGAAGTCGAGAGTGTGCGGGTACTGCGGGGCCCCGGCCC CATACG CTACTGCCTGTGGCCTCGACGTCTGCATTTACCACACCCACTTCCACCAGCATTGTCCAGTCACAATCTGGTGTGGCCATCCAGCGGGTTCTGGTTCTTGTAGTGAGTGCAAATCCCCTGTAGGGAAAGGCACAAGCCCTTTAGACGAGGTGCTGGAACAAGTCCCGTATAAGCCCCCACGGACCGTTATCATGCATGTGGAGCAGGGTCTCACCCCCCTTGATCCAGGTAGATACCAAACTCGCCGCGGACTAGTCTCTGTCAGGCGTGGAATTAGGGGAAATGAAGTTGAACTACCAGACGGTGATTATGCTAGCACCGCCTTGCTCCCTACCTGCAAAGAGATCAACATGGTCGCTGTCGCTTCCAGTGTATTGCGCAGCAGGTTCATCATCGGCCCACCCGGTGCTGGGAAAACATACTGGCTCCTTCAACAGGTCCAGGATGGTGATGTTATTTACACACCAACTCACCAGACCATGCTTGACATGATTAGGGCTTTGGGGACGTGCCGGTTCAACGTCCCGGCAGGCACAACGCTGCAATTCCCCGTCCCCTCCCGCACCGGTCCGTGGGTTCGCATCCTAGCCGGCGGTTGGTGTCCTGGCAAGAATTCCTTCCTTGATGAAGCAGCGTATTGCAATCACCTTGATGTTTTGAGGCTTCTTAGTAAAACTACCCTCACCTGTCTAGGAGACTTCAAGCAACTCCACCCAGTGGGTTTTGATTCTCATTGCTATGTTTTTGACATCATGCCTCAAACTCAACTGAAGACCATCTGGAGGTTTGGACAGAATATCTGTGATGCCATTCAGCCAGATTACAGGGACAAACTCATGTCCATGGTCAACACAACCCGTGTGACCTACGTGGAAAAACCTGTCAGGTATGGGCAGGTCCTCACCCCCTACCACAGGGACCGAGAGGACGACGCCATCACTATTGACTCCAGTCAAGGCGCC 7 ACATTCGATGTGGTCACATTGCATTTGCCCACTAAAGATTCACTCAACAGGCAAAGAGCCCTTGTTGCCATCACCAGGGCAAGACACGCTATCTTTGTGTATGACCCACACAGGCAGCTGCAGGGCTTGTTTGATCTTCCTGCAAAAGGCACACCCGTCAACCTCGCAGTGCACCGCGACGGGCAGCTGATCGTGCTGGATAGGAATAACAAAGAATGCACGGTTGCTCAGGCCCTAGGCAACGGGGATAAATTTAGGGCCACGGATAAGCGTGTTGTAGATTCTCTCCGCGCCATTTGTGCTGATCTAGAAGGGTCGAGCTCTCCGCTCCCCAAGGTCGCACACAACTTGGGATTTTATTTCTCACCTGATTTAACACAGTTTGCTAAACTCCCAGTAGAACTTGCACCTCACTGGCCCGTGGTGACAACCCAGAACAATGAAAAGTGGCCAGATCGGCTGGTTGCCAGCCTTCGCCCTATCCATAAATACAGCCGCGCGTGCATCGGTGCCGGCTATATGGTGGGCCCTTCGGTGTTTCTAGGCACTCCTGGGGTCGTGTCATACTATCTCACAAAATTTGTTAAGGGTGAAGCTCAATTGCTTCCGGAGACGGTTTTCAGCACCGGCCGAATTGAGGTAGACTGCCGGGAATATCTTGATGATCGGGAGCGAGAAGTTGCTGCGTCCCTCCCACACGCTTTCATTGGCGACGTCAAAGGCACTACCGTTGGAGGATGTCATCATGTCACCTCCAGATACCTCCCGCGTGTCCTTCCCAAGGAATCAGTTGCGGTGGTCGGGGTTTCAAGCCCCGGAAAAGCCGCGAAAGCATTGTGCACACTGACAGATGTGTACCTCCCAGATCTTGAAGCCTATCTCCACCCGGAGACCCAGTCCAAGTGCTGGAAAATGACGTTGGACTTCAAAGAAGTTCGACTAATGGTCTGGAAAGACAAAACAGCCTATTTCCAACTTGAAGGTCGCTATTTCACTTGGTATCAGCTTGCCAGCTATGCCTCGTACATCCGTGTTCCTGTCAACTCTACGGTGTACTTGGACCCCTGCATGGGCCCCGCCCTTTGCAACAGGAGAGTCGTCGGGTCCACCCACTGGGGGGCTGACCTCGCGGTCACCCCTTATGATTACGGCGCTAAAATTATCCTGTCTAGCGCGTACCATGGTGAAATGCCCCCCGGATATAAAATTCTGGCGTGCGCGGAGTTCTCGTTGGATGACCCAGTTAAGTACAAACATACCTGGGGGTTTGAATCGGATACAGCGTATCTGTATGAGTTCACTGGAAACGGTGAGGACTGGGAGGACTACAATGATGCGTTTCGTGCGCGCCAGGAAGGGAAAATTTACAAGGCCACTGCCACCAGCTTGAAGTTTTATTTTCCCCCGGGCCCTGTCATTGAACCAACTTTAGGCCTGAATTGAAATGAAATGGGGTCCATGCAAAGCCTTTTTGACAAAATTGGCCAACTTTTTGTGGATGCTTTCACGGAGTTCTTGGTGTCCATTGTTGATATCATTATATTCTTGGCCATTTTGTTTGGCTTCACCATCGCCGGTTGGCTGGTGGTCTTTTGCATCAGATTGGTTTGCTCCGCGATACTCCGTACGCGCCCTGCCATTCACTCTGAGCAATTACAGAAGATCTTATGAGGCCTTTCTTTCCCAGTGCCAAGTGGACATTCCCACCTGGGGAACTAAACATCCTTTGGGGATACTTTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCGCGTCGAATATACCGCATCATGGAAAAATCAGGGCAGGCTGCCTGGAAACAGGTGGTGAGCGAGGCTACGCTGTCTCGCATTAGTAGTTTGGATGTGGTGGCTCATTTTCAGCATCTAGCCGCCATTGAAGCCGAGACCTGTAAATATTTGGCCTCCCGGCTGCCCATGCTACACAACCTGCGCATGACAGGTTCAAATGTAACCATAGTGTATAACAGCACTTTGAATCAGGTGTTTGCTATTTTTCCAACCCCTGGTTCCCGGCCAAAGCTTCATGATTTTCAGCAATGGTTAATAGCTGTACA 8 TTCCTCTATATTTTCCTCTGTTGCAGCTTCTTGTACTCTTTTTGTTGTGCTGTGGTTGCGGGTTCCAATACTACGTACTGTTTTTGGTTTCCGCTGGTTAGGGGCAATTTTTCTTTCGAACTCACAGTGAATTACACGGTGTGTCTACCTTGCCTCACCCGGCAAGCAGCCACAGAGATCTACGAACCCGGTAGGTCTCTTTGGTGCAGGATAGGGTATGACCGATGTGAGGAGGATGATCATGACGAGCTAGGGTTTATGGTACCGCCTGGCCTCTCCAGCGAAGGCCACTTGACTAGTGTTTACGCCTGGTTGGCGTTCTTGTCCTTCAGTTACACGGCCCAGTTCCATCCCGAGGTATTCGGGATAGGGAATGTGAGTCGAGTTTATGTTGACATCAAACATCAACTCATCTGCGCCGAACATGACGGGCAGAACACCACCTTGCCTCGTCATGACAACATTTCAGCCGTGTTTCAGACCTATTACCAACATCAAGTCGACGGCGGCAATTGGTTTCACCTAGAATGGCTTCGTCCCTTCTTTTCCTCGTGGTTGGTTTTAAATGTCTCTTGGTTTCTCAGGCGTTCGCCTGCAAACCATGTTTCAGTTCGAGTCTTGCAGATATTAAGACCAACACCACCGCAGCGGCAGGCTTTGCTGTCCTCCAAGACATCAGTTGCCTTAGGCATCGCGACTCGGCCTCTGAGGCGATTCGCAAAATCCCTCAGTGCCGTACGGCGATAGGGACACCCGTGTATGTTACCATCACAGCCAATGTGACAGATGAGAATTATTTACATTCTTCTGATCTCCTCATGCTTTCTTCTTGCCTTTTCTATGCTTCTGAGATGAGTGAAAAGGGATTTAAGGTGGTATTTGGCAATGTGTCAGGCATCGTGGCTGTGTGTGTCAATTTTACCAGCTACGTCCAACATGTCAAGGAGTTTACCCAACGCTCCCTGGTGGTCGACCATGTGCGGTTGCTCCATTTCATGACACCTGAGACCATGAGGTGGGCAACTGTTTTAGCCTGTCTTTTTGCCATTCTGTTGGCAATTTGAATGTTTGAGTATGTTGGAGAAATGCTTGACCGCGGGCTGTTGCTCGCGATTGCTTTCTTTGTGGTGTATCGTGCCGTTCTGTTTTGCTGCGCTCGCCAACGCCAGCGGCAACAGCAGCTCCCATCTACAGCTGATTTACAACTTGACGCTATGTGAGCTGAATGGCACAGATTGGCTAGCTAACAAATTTGATTGGGCAGTGGAGAGTTTTGTCATCTTTCCTGTTTTGACTCACATTGTCTCCTACGGTGCCCTCACCACCAGTCATTTCCTTGACACGGTTGCCTTGACCACCGTGTCTACCGCCGGATTTGTTCACGGGCGGTATGTCCTAAGCAGCATCTACGCGGTCTGTGCCCTGGCTGCGTTGCTTTGCTTCATCATCAGGTTTGCAAAGAATTGCATGTCCTGGCGCTATGCGTGCACCAGATACACCAATTTTCTTCTGGATACCAAGGGTAGACTCTATCGCTGGCGGTCGCCTGTCATCATAGAGAAAAGGGGCAAAGTTGAGGTCGTAGGTCATCTAATCGACCTAAAAAAAGTTGTGCTTGATGGTTCCGTGGCAACCCCTATAACCAGAGTTTCAGCGGAACAATGGGGTCGTCCTTAGATGACTTCTGTCATGATAGCACGGCTCCACAAAAGGTGCTTTTGGCTTTTTCCATTACCTACACACCAGTGATGATATATGCCCTGAAGGTGAGTCGCGGCCGACTGCTGGGGCTTCTGCACCTTTTGATCTTCCTAAATTGTGCCTTCACTTTCGGGTACATGACTTTCGTACACTTTAAGAGTACAAATAAGGTCGCGCTCACTATGGGAGCAGTAGTTGCACTCCTTTGGGGGGTGTACTCAGCCATAGAAACCTGGAAATTCATCACCTCCAGATGCCGTTTGTGCTTGCTAGGCCGCAAGTACATCCTGGCCCCTGCCCACCACGTTGAAAGTGCCGCAGGCTTTCATTCGATTGCGGCAAATGATAACCACGCATTTGTCGTCCGGCGTCCCGGCTCCACTACGGTCAACGGCACATTGGTGCCCGGGTTGAAAAGCCTCGTGTTGGGTGGCAGAAAAGCTGTTAAACAGGGAGTGGTAAACCTTGTCAAATATGCCAAATAACAACGGCAAGCAGCAGAAGAGAAAGAAGGGGGATGGCCAGCCAGTCAATCAGCTGTGCCAGATGCTGGGCAAGATCATTGCTCAGCAAAATCAGTCCAGAGGTAAGGGACCGGGGAAGAAAAACAAAAAGAAACAACCGGAGAAGCCCCATTTTCCTCTAGCGACTGAAGATGATGTCAGACATCACTTTACCCCTAGTGAGCGGCAATTGTGTCTGTCGTCAATCCAGACCGCCTTCAATCAAGGCGCTGGGACTTGCACCCTGTCAGATTCGGGGAGGATAAGTTACACTGTGGAGTTCAGTCTGCCTACGCATCATACCGTGCGCCTTATTCGTGTCACGGCTTCACCCACGGCATGATGGGCTGGCATTCTTGAGGCACCCCAGTGCTTAAATTGGAAGAATGTGTGGTGAATGGCACTGATTGATATTGTGCCTCTAAGTCACCTATTCAATTAGGGCGACCGTGTGGGGGTAAGATTTAATTGGCGAGAACCATGCGGCCGAAATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0071] [Example 2]

[0072] CSNA11-Lineage1 sequence acquisition and CSNA11-Lineage1 chimeric virus (CSNA11-L1 chimeric virus) production

[0074] 2-1. CSNA11-Lineage1 Virus isolation and sequence acquisition

[0075] Lineage 1 of the North American PRRS virus genotype is a field strain that has recently been spreading rapidly in Korea, and since strains such as NADC-30 and MN-184 belonging to the same Lineage 1 are known to be highly virulent, a new vaccine is required against it.

[0076] Accordingly, the inventors isolated highly pathogenic PRRS Lineage1 virus from PAM (alveolar macrophages) and identified the structural protein coding region (total 3188 bp) of Table 2 below to obtain its nucleotide sequence (ORF2~ORF7, SEQ ID NOs 9 to 14).

[0078] Sequence number nucleotide sequence 9 ATGAAATGGGGGCCCTGCAGAGCCTGTTTGACAAAATCGGCCAACTGTTTGTGGACGCTTTCACGGAGTTCTTGGTTTCTGTTGTTGATATCATCATATTTTTGGCCATTTTGTTCGGCTTCACCATCGCCGGATGGTTGGTGGTATTTTGCATCAGAATGGTTTTCTCCGCGATACTCCGTGCGCGCTCTGCCGTTCACCCTGAGCAATTACAGAAGATCCTATGAGGCTTACCTTTCCCAGTGCCAGGCAGACATCCCTTCCTGGGGCACAAAACACCCCTTGGGAATGTTTTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCACGACGCATGTACCGCACCATGGAGAAAGCAGGTCAGGCAGCTTGGAAACAGGTAGTTAGCGAAGCCACGCTGTCTCGCATCAGTGGCTTGGATGTGGTAGCTCATTTCCAGCACCTTGCTGCCATAGAAGCCGAGACTTGTAAATATTTGGCCTCTCGGCTTCCTATGCTACATAACCTGCGTATGACAGGGTCAAATGTAACCATAAAATATAATAGTACCTCTGGCCAGGTATTTGCTATCTTCCCAACTCCAGGCTCCCGGCCCAAGCTTCATGATTTTCAGCAATGGTTAATAGCTGTGCACTCCTCCATATTCTCCTCTGTTGCAGCTTCTTGCACCCTATTCATTGTGCTGTGGCTACGAGTGCCAATTCTACGTACTGTTTTTGGTTTCCGTTGGTTAGGGGCAATTTTTCCTTCGAGCTCATGGTGA 10 ATGGTTAATAGCTGTGCACTCCTCCATATTCTCCTCTGTTGCAGCTTCTTGCACCCTATTCATTGTGCTGTGGCTACGAGTGCCAATTCTACGTACTGTTTTTGGTTTCCGTTGGTTAGGGGCAATTTTTCCTTCGAGCTCATGGTGAATTATACTGTGTGCCCACCCTGCCTCACCCGGCAGGCGGTCGCTCAGATCCTCGAACCCGGTAGATCTTTGTGGTGTAGGATAGGGTCTGATCGATGCGAGGAAAGCGATCATGATGAGCTGGAATTTGCTGTGCCGCCCGGCCTTTCTCAAGAGGGTCATCTGACCAGTGTCTATGCTTGGTTAGCGTTTTTGTCCTTCAGCTACACAGCACAGTTTCATCCCGAAGTATTCGGGATAGGGAATGTGAGCCAGGTTTATGTTGATATCAAGAAGCAACTCATCTGTGCTGACCATGACGGACAGAACGCCACCTTGCCCAGCCATGACAACATTACGGCCGTGTATCAGACTTATTACCAGCATCAAGTCGATGGGGGAAATTGGTTTCACCTGGAATGGCTGCGTCCCTTCTTTTCCTCTTGGTTGGTTTTGAACGTCTCATGGTTTCTCAGGCGTTTGCCTGCAAGCCGTGTTTCAGTTCGAGTCTTTCAGACATTGAGACCAACACCACCAGCGCAGCAAGTTCCGTTGTCCTCCAAGACATCAGCTGTCTTAGGCATGGCAACCAGGCCCGTGCGACAATTCGCAAAACTCCTCAATGTCGCACGGCGATAG 11 ATGGCTGCGTCCCTTCTTTTCCTCTTGGTTGGTTTTGAACGTCTCATGGTTTCTCAGGCGTTTGCCTGCAAGCCGTGTTTCAGTTCGAGTCTTTCAGACATTGAGACCAACACCACCAGCGCAGCAAGTTCCGTTGTCCTCCAAGACATCAGCTGTCTTAGGCATGGCAACCAGGCCCGTGCGACAATTCGCAAAACTCCTCAATGTCGCACGGCGATAGGAACACCAGTCTATATCACTATTACAGCCAACGTGACGGACGAGAATTATCTGCATTCCTCCGACCTTCTCATGCTCTCTTCTTGCCTTTTCTATGCCTCTGAGATGAGTGAAAAGGGATTTAAGGTGATATTTGGCAATGTGTCAGGCGTTGTGGCTGTGTGTATCAATTTTACAAGCTATGTCCAACATGTCAAGGAGTTCACCCGACAATCCTTAGTGGTCGACCATGTGCGGCTGCTTCACTTCATGACACCTGAAACTATGCGGTGGGCGACCGTTTTAGCCTGTCTTTTTGCCATTCTGTTGGCAATTTGA 12 ATGTTGGGGAAATGCTTGACCGCGGGTTGTTGCTCGCAATTGCTTTTTTTGTGGTGTATCGTGCCATTCTGGTTTGCTGTGCTCGTCAACGCCGGCAACACCAGCAGCTCCCATCTACAGTTGATTTATAACCTGACGATATGTGAGCTGAATGGTACAGATTGGCTAGATAAACATTTTTCCTGGGCAGTGGAGACTTTCGTCATTTTTCCTGCGTTGACTCACATTGTCTCTTACGGCGCCCTTACCACTAGCCATTTCCTTGACATGGTTGGCCTGATCACTGTGTCTGCCGCCGGATATTTTCACGAGCGGTATGTGTTGAGTAGCATCTATGCTGTCTGTGCCCTGGCCGCGTTGGTTTGCTTCATCATCAGGCTGTCAAAAAATTGCATGTCCTGGCGCTATTCATGCACCAGATATACCAATTTTCTTCTGGATACTAAGGGCAATCTTTACCGCTGGCGGTCACCTGTCATCATAGAGAAAGGGGGTAAGGTCGATGTTGGGAATCACTTAATCGACCTCAAAAGAGTTGTGCTTGATGGTTCCGCAGCAACCCCTATAACCAAGATTTCAGCTGAGCAATGGGGTCGTCTATAG 13 ATGGGGTCGTCTATAGACGACTTTTGCAATGACAGCACGGCTGTACAAAAGGTGCTATTGGCGTTTTCTATCACCTACACGCCAATAATGATATATGCCCTAAAGGTAAGTCGTGGTCGACTGCTGGGGCTCTTGCACCTCTTAATTTTCCTGAATTGTGCTTTCACTTTTGGGTACATGACATTTGTTCATTTTCAGAGTACAAACAAAGTCGCACTTACTATGGGGGCAGTAGTCGCTCTCCTCTGGGGGGTGTATTCAGCCATTGAAACCTGGAGATTCATCACCTCCAGATGCCGGTTGTGCTTGCTAGGCCGCAAGTACATTCTGGCCCCTGCCCACCACGTTGAAAGTGCCGCAGGCTTTCATCCGATAACGGCAAGTGATAACCACGCATTTGTCGTCCGGCGTCCCGGCTCCACTACGGTCAACGGCACATTGGTGCCCGGGTTGAAAAGCCTCGTGTTGGGTGGCAGAAGAGCTGTTAAACAAGGAGTGGTGAACCTTGTCAAATATGCCAAATAA 14 ATGCCAAATAACAACGGCAGACAGCAAAATAAAAAGAAGGGGGGTGGCCAGCCAGTCAATCAGCTGTGCCAGATGTTGGGTAAGATTATTGCCCAACAGAATCAGTCCAGAGGCAAGGGACCGGGGAAGAGGAACAAGAATAAAAACCCGGAGAAGCCCCACTTTCCTCTAGCGACTGAAGATGACGTCAGGCATCACTTCACCCCCAGTGAGCGACAATTGTGTCTGTCGTCAATCCGGACTGCCTTTAATCAAGGCGCTGGAACTTGCACCCTGTCAGACTCAGGGAGGATAAGTTACACCGTGGAGTTTAGTTTGCCTACTCAGCATACTGTGCGCTTAATTCGTGTCACAGCGTCACCTTCAGCCTGA

[0079] 2-2. CSNA11-Lineage1 키메릭 바이러스 제작

[0080] A portion of the nucleotide sequence of SEQ ID NO. 8 of the CSNA11 infectious clone prepared in Example 1 is PRRS Lineage1 virus A chimeric virus was produced by replacing the ORF5 gene (sequence number 12), but no virus was produced. This was because the PRRS Lineage 1 virus has a viral gene sequence that is not adapted to MARC-145 cells, and it was determined that the virus recovery efficiency was significantly reduced when producing the virus using an infectious clone, so it was produced using a different method.

[0081] The ORF5 ectodomain of the PRRSV gene contains a major neutralizing epitope and major regions associated with cross-neutralization. In Table 3, to construct a CSNA11-Lineage1 chimeric virus infectious clone, at least 13 nucleotides were substituted in the CSNA11 ectodomain nucleotide sequence (Sequence No. 15) to substitute 8 amino acids in the Lineage 1 ORF5 ectodomain (Sequence No. 16). As a result, the nucleotide homology between the ectodomain of the CSNA11-Lineage1 chimeric virus infectious clone and the ORF5 ectodomain of the CSNA11 virus was 86%, which was higher than the homology with the Lineage 1 virus ectodomain, which was 82.7%. This was designed to be as close as possible to the CSNA11 ORF5 ectodomain gene sequence used to construct the virus using the infectious clone in Example 1.

[0083] 서열번호 염기서열 15 AGCGGCAACAGCAGCTCCCATCTACAGCTGATTTACAACTTGACGCTATGTGAGCTGAATGGCACAGATTGGCTAGCTAACAAATTTGATTGG 16 G GC AA CA C CAGCAGCTCCCATCTACAGCTGATTTACAACTTGACG A T C TGTGAGCTGAATGGCACAGATTGGCT T G A TAA AC A T TTT TC TTGG

[0084] The above ectodomain nucleotide sequence substitution was performed using the QuikChange II XL site-directed mutagenesis kit (Agilent, Palo Alto, CA, USA) according to the product protocol. As a result of transfecting MARC-145 cells with the plasmid using ViaFect transfection reagent (Promega, Madison, WA, USA) as an infectious clone of the CSNA11-Lineage1 chimeric virus produced in this example, a CSNA11-L1 chimeric virus identical to the Lineage 1 ectodomain amino acid sequence (Sequence No. 21) was produced.

[0086] ORF5 gene analysis of the above-mentioned virus up to 9 cell passages revealed no mutations toward the field isolate CSNA11 virus gene, and finally, the replication capacity of the CSNA11-Lineage1 chimeric virus was 10 6.69 TCID 50 Confirmed as / ml, and titer at 1 passage (10 4.50 TCID 50 It can be seen that it is usable as a vaccine strain as it has increased compared to / ml).

[0088] [Example 3]

[0089] Production of CSNA11-L1-N44S chimeric virus (Hypo-glycosylated CSNA11-L1 chimeric virus)

[0091] To improve the formation of neutralizing antibodies against the PRRS virus, a CSNA11-L1-N44S chimeric virus with an improved neutralizing antibody production rate was prepared by exposing a glycosylated neutralizing epitope in the ORF5 region of the PRRS virus.

[0092] First, the 44th amino acid Asparagine (N) of the ORF5 of the PRRS virus is an important amino acid for viral replication, and it is known that if the said amino acid Asparagine (N) is replaced with another amino acid, the virus is not produced (U.S. Patent Publication No. 20080233083). However, the inventors confirmed as follows that when the 44th amino acid in the amino acid sequence of the ORF5 of the PRRS virus (Sequence No. 18) is changed to Serine (S), the virus is produced without structural modification of the virus.

[0093] Specifically, an infectious clone was constructed using a sequence (Sequence No. 17) in which the 38th base, Adenosine (A), in the ORF5 ectodomain of the CSNA11-L1 virus was substituted with Guanine (G). Using the above-mentioned infectious clone, a CSNA11-L1-N44S chimeric virus was constructed having an amino acid sequence (Sequence No. 19) in which the 44th amino acid, Asparagine (N), in the ORF5 amino acid sequence (Sequence No. 18) of the CSNA11-L1 virus was substituted with Serine (S).

[0095] Sequence number nucleotide sequence 17 GGCAACACCAGCAGCTCCCATCTACAGCTGATTTACAGCTTGACGATCTGTGAGCTGAATGGCACAGATTGGCTTGATAAACATTTTTCTTGG

[0096] Sequence number amino acid sequence 18 MLEKCLTAGCCSRLLSLWCIVPFCFAALANA GNTSSHLQLIYNLTICELNGTDWLDKHFSW AVESFVIFPVLTHIVSYGALTTSHFLDTVALTTVSTAGFVHGRYVLSSIYAVCALAALLCFIIRFAKNCMSWRYACTRYTNFLLDTKGRLYRWRSPVIIEKRGKVEVVGHLIDLKKVVLDGSVATPITRVSAEQWGRP 19 MLEKCLTAGCCSRLLSLWCIVPFCFAALANA GNTSSHLQLIYSLTICELNGTDWLDKHFSW AVESFVIFPVLTHIVSYGALTTSHFLDTVALTTVSTAGFVHGRYVLSSIYAVCALAALLCFIIRFAKNCMSWRYACTRYTNFLLDTKGRLYRWRSPVIIEKRGKVEVVGHLIDLKKVVLDGSVATPITRVSAEQWGRP 20 SGNSSSSHLQLIYNLTLCELNGTDWLANKFDW 21 GNT SSSHLQLIYNLT I CELNGTDWL DKH F S W

[0097] Specifically, an infectious clone of the CSNA11-L1-N44S chimeric virus was produced by following the protocol using the QuikChange II XL site-directed mutagenesis kit (Agilent, Palo Alto, CA, USA). Subsequently, the produced infectious clone was transfected into MARC-145 cells using ViaFect transfection reagent (Promega, Madison, WA, USA), and the CSNA11-L1-N44S chimeric virus was obtained.

[0098] Sequence analysis of the ORF5 gene after 6 passages using the above-described chimeric virus confirmed that it is a gene of the CSNA11-L1 chimeric virus and no back mutations were observed. Additionally, it was confirmed that the deglycosylation sequence was maintained during the 6 passages, and the final viral replication rate was 10 6.75 TCID 50 In / ml, titer at 1 passage (10 4.13 TCID 50 It was confirmed that it can be used as a vaccine strain as it increased compared to / ml).

[0100] [Example 4]

[0101] In-vitro characterization of rescued chimeric virus

[0103] 4-1. Indirect immunofluorescence assay (IFA)

[0104] Indirect immunofluorescence assay (IFA) was performed to confirm the replication of CSNA11-L1 chimeric virus and CSNA11-L1-N44S chimeric virus in MARC-145 and PAM (Pulmonary Alveolar Macrophage). MARC-145 cells and PAM cells were seeded on culture slides for IFA one day prior to virus inoculation. After inoculating each cell with the chimeric virus and staining with IFA antibodies 48 hours later, as shown in Figures 1 and 2, it was confirmed that protein expression of the PRRS virus in the cells occurred normally for both types of chimeric viruses, CSNA11-L1 and CSNA11-L1-N44S.

[0106] 4-2. Using PAM in vitro virulence test

[0107] Pulmonary Alveolar Macrophages (PAMs) are target cells for the PRRS virus; it is known that attenuated strains grow well on MARC-145 but not on PAMs. It is known that if the viral replication rate on MARC-145 is more than 100 times higher than on PAMs, the virus can be evaluated as having been attenuated in the pig (Kwon et al., 2006). As shown in Table 6 below, the titers on PAMs for two types of chimeric viruses, including CSNA-11, were observed to be 390 to 4780 times lower than the viral replication rate on MARC-145s. In particular, a larger difference was observed in the two types of chimeric viruses (2400–4780 times) compared to the low-pathogenicity wild-type isolate (CSNA11, 390 times), leading to the conclusion that they are safer in pigs as they are more attenuated than the low-pathogenicity CSNA11 wild-type virus.

[0109] Types of viruses logTCID 50 / ml PAM MARC-145 CSNA11 (wildtype) 4.31 6.90 CSNA11-L1 chimeric virus 3.00 6.68 CSNA11-L1-N44S chimeric virus 3.38 6.75

[0110] 4-3. In vitro stability test

[0111] After analyzing the ORF5 gene following 15 additional passages to check for genetic changes in the CSNA11-L1 chimeric virus, it was confirmed that there were no mutations.

[0113] [Example 5]

[0114] Safety and Immunogenicity Evaluation of CSNA11-L1 Chimeric Virus

[0116] Twelve 4-week-old pigs confirmed to be negative for PRRSV were classified as shown in Table 7 below. High doses (10 doses or more) of the vaccine candidate were administered to 4-week-old piglets, and vaccine safety and immunogenicity were assessed up to 42 days after vaccination. The study consisted of a single vaccination and eight blood draws (0, 3, 7, 10, 14, 21, 28, and 42 dpv). The safety and immunogenicity of the vaccine candidate were analyzed using the collected blood samples through viremia level, ELISA antibody titer, neutralizing antibody titer, and Porcine IFN-γ secreting cell scoring. Statistical analysis of the results was performed using the 2-way ANOVA method.

[0118] army Inoculation solution (0 DPI) Dusu Negative control group None 4 heads positive control group wild-type virus (CSNA11)(10 6.0 TCID 50 / 2 mL / dose, IM) 4 heads Vaccine group CSNA11-L1 chimeric virus(10 6.0 TCID 50 / 2 mL / dose, IM) 4 heads Total 12 heads

[0119] As shown in Figure 3, when body temperature was measured from 0 to 14 DPV after virus inoculation, no significant difference was observed between the negative control group, the CSNA11 wild-type virus, and the groups inoculated with the CSNA11-L1 chimeric virus.

[0120] As shown in Figure 4, the ADWG analysis results of 0 to 42 DPVs showed that the ADWG of the CSNA11-L1 chimeric virus inoculated group and the CSNA11 wild-type virus inoculated group were higher than that of the negative control group, but no statistically significant difference was observed.

[0121] As shown in Figure 5, these are the results of analyzing the titers of North American PRRSV contained in serum using serum collected at different intervals after vaccination. Serum viremia was confirmed starting from day 3 after vaccination in the group inoculated with the CSNA11-L1 chimeric virus and the group inoculated with the CSNA11 wild-type virus. The serum viremia in the group inoculated with the CSNA11 wild-type virus reached its highest level on day 10 after vaccination, at 10 2.7 TCID 50It showed / ml and gradually decreased until the end of the experiment. Serum viremia in the group inoculated with the CSNA11-L1 chimeric virus was 10 on day 7 after vaccination. 1.27 TCID 50 The values ​​were shown as / ml, and were not detected starting from the 14th day after vaccination. In the CSNA11 wild-type virus inoculated group, the virus persisted in the pig blood for up to 28 days, whereas in the CSNA11-L1 chimeric virus inoculated group, it was present weakly in the blood for up to 10 days and completely disappeared from the blood on the 14th day. Significantly lower viremia levels were confirmed compared to the CSNA11 wild-type virus inoculated group at both 10 and 14 days after vaccination (p<0.001).

[0122] As shown in Figure 6, the results of Porcine IFN-γ ELISpot analysis were performed using PBMCs isolated from porcine whole blood on days 21 and 42 after virus inoculation. The isolated PBMCs were sensitized with the CSNA11 wild-type virus. The IFN-γ recall response secreted by PBMCs in response to the antigen was analyzed when the PBMCs collected after vaccination were re-exposed to the corresponding antigen. The number of ELISpot plate spots was analyzed using an AID ISPOT (ELR08IFL) ELISpot reader. In the group inoculated with the CSNA11-L1 chimeric virus, an increasing trend in the number of IFN-γ secreting cells was observed up to day 42 after inoculation. In contrast, in the group inoculated with the CSNA11 wild-type virus (wild-type, WT), a decrease in the number of IFN-γ secreting cells was observed from day 21 to day 42 after virus inoculation. In particular, when comparing the cellular immune responsiveness between the two groups 42 days after vaccination, it was confirmed that the CSNA11-L1 chimeric virus inoculated group was significantly higher (p<0.01).

[0123] As shown in Figure 7, the results of PRRS antibody ELISA were performed using serum collected at different time intervals. The analysis was conducted according to the protocol of the IDEXX PRRS X3 Ab Test (IDEXX Laboratories, USA) kit. SP antibodies against the PRRS virus began to be produced starting from day 7 after inoculation with the CSNA11-L1 chimeric virus and the CSNA11 wild-type virus, and positive levels of antibodies were detected on the antibody kit after day 10. Although it was confirmed that slightly higher levels of antibodies were produced in the CSNA11 wild-type virus inoculation group between days 10 and 21 compared to the group inoculated with the genetically engineered CSNA11-L1 chimeric virus, no significant difference in SP antibody production was found between the groups. At day 42 after inoculation, it was confirmed that similar levels of SP antibodies against the PRRS virus were produced in both groups.

[0125] [Example 6]

[0126] Efficacy evaluation against CSNA11-L1-N44S chimeric virus

[0128] Eight 4-week-old pigs confirmed to be negative for PRRSV were classified as shown in Table 8 below. 4-week-old piglets were vaccinated with an overdose (10 doses) of the CSNA11-L1-N44S chimeric virus, and vaccine safety and immunogenicity were confirmed up to 42 days after vaccination. Additionally, 42 days after vaccination, a challenge was performed with the PRRS North American lineage 1 wild-type virus, and euthanasia and necropsy were performed 19 days later. The PRRS North American lineage 1 wild-type virus used for the challenge was obtained by isolating the highly pathogenic North American field strain (Lineage 1, KU-PRRSV-2020-002) from PAM (alveolar macrophages) in Example 2. The study involved a single vaccination, a single challenge inoculation, and 14 blood draws (0, 1, 3, 7, 10, 14, 21, 28, 42 dpv, 1, 3, 7, 14, 19 dpc), and clinical symptoms were observed up to 19 dpc. The safety and immunogenicity of the vaccine candidate were analyzed using viremia levels, ELISA antibody titers, and neutralizing antibody titers based on the collected blood samples. The Mann-Whitney test, a non-parametric test, was used for statistical analysis of efficacy evaluation.

[0130] army Inoculation solution (0 DPI) Challenge solution (42 DPI, 0 DPC) Dusu Negative control group None Lineage 1 wild-type (10 5.0 TCID 50 / 1mL / dose, IM) 4 heads Vaccine group CSNA11-L1-N44S chimeric virus(10 5.5 TCID 50 / 1 mL / dose, IM) Lineage 1 wild-type (10 5.0 TCID 50 / 1mL / dose, IM) 4 heads Total 8 heads

[0131] It was determined that the vaccine was non-pathogenic and safe because there were no significant differences between the vaccine group and the negative control group in clinical symptoms, body temperature, ADWG (average daily weight gain), viremia level, etc., for 42 days after administering an overdose (10 doses) of the vaccine.

[0132] Analysis of clinical symptom scores after challenge showed that no clinical symptoms were observed in either the CSNA11-L1-N44S chimeric virus inoculated group or the negative control group at time points excluding 14 DPC (day post-challenge); however, at 14 DPC, the clinical symptom scores (activity, depression) of the CSNA11-L1-N44S chimeric virus inoculated group were significantly lower than those of the negative control group (p=0.029).

[0133] As shown in Figure 8, at all time points observed after challenge, the body temperature of the CSNA11-L1-N44S chimeric virus inoculated group was lower than that of the negative control group. At 3 DPC, the body temperature of the CSNA11-L1-N44S chimeric virus inoculated group was significantly lower than that of the negative control group (p=0.029).

[0134] As shown in Figure 9, the ADWG analysis of 0 to 19 DPCs showed that the ADWG of the CSNA11-L1-N44S chimeric virus inoculated group was higher than that of the negative control group, but no significant difference was observed (p=0.083).

[0135] As shown in Figure 10, the persistence of the field virus in the blood and the distribution of viremia content in the blood after challenge were investigated. The CSNA11-L1-N44S chimeric virus vaccine group showed significantly lower distributions in DPCs 1 and 3 compared to the challenge negative control group, and in DPC 7, the viremia level of the CSNA11-L1-N44S chimeric virus vaccine group was significantly lower than that of the negative control group (p<0.001). In addition, it was confirmed that the field pathogenic virus infection disappeared from the blood earlier compared to the negative control group with the CSNA11-L1-N44S chimeric virus vaccine. This result indicates that when challenged with the field virus, blood viremia is reduced in the CSNA11-L1-N44S chimeric virus inoculated group compared to the negative control group, and it demonstrates the effect of rapidly reducing the field-infected virus when used as a live vaccine on farms.

[0136] As shown in Figure 11, serum collected at each efficacy evaluation period was analyzed according to the protocol of the IDEXX PRRS X3 Ab Test (IDEXX Laboratories, Inc., Columbus, OH, USA) kit to confirm PRRS antibodies. Since ELISA antibodies increased in all groups vaccinated against the CSNA11-L1-N44S chimeric virus 35 days after vaccination, it can be concluded that the attenuated vaccine strain is immunogenic.

[0137] As shown in Figure 12, the neutralizing antibody titers were measured, and the CSNA11-L1-N44S chimeric virus inoculated group showed a high neutralizing antibody titer of 1:23 at 42 DPI, and neutralizing antibody titers of 1:181 to 1:304 were observed after challenge. The neutralizing antibody titer of the negative control group after challenge was < 1:4, which was significantly lower than that of the CSNA11-L1-N44S chimeric virus inoculated group (p < 0.05).

[0138] A necropsy was performed 19 days after challenge to collect lung tissue and tracheobronchian lymph nodes (TBLN) from each experimental group. As a result of measuring the residual viral content in the tissues after field challenge, no significant difference was observed between the CSNA11-L1-N44S chimeric virus inoculated group and the negative control group. However, as shown in Figure 13, the megapulmonary lesion analysis results showed that the megapulmonary lesion index of the CSNA11-L1-N44S chimeric virus inoculated group (mean 10.13, standard deviation 6.11) was significantly lower than that of the negative control group (mean 18.41, standard deviation 3.36) (p=0.029). In addition, the micropulmonary lesion analysis results showed that the micropulmonary lesion index of the CSNA11-L1-N44S chimeric virus inoculated group (mean 1.3, standard deviation 0.32) was significantly lower than that of the negative control group (mean 2.18, standard deviation 0.75) (p=0.007).

Claims

Claim 1 delete Claim 2 A vector for producing an attenuated porcine reproductive and respiratory syndrome chimeric virus, characterized in that, in a vector for producing a porcine reproductive and respiratory syndrome virus strain CSNA11 containing the nucleotide sequence of SEQ ID NO. 1, the nucleotide sequence of SEQ ID NO. 15 corresponding to the nucleotide sequence of the ORF5 ectododomain is changed to the nucleotide sequence of SEQ ID NO. 16 or 17. Claim 3 An attenuated porcine reproductive and respiratory syndrome chimeric virus encoded by a sequence in which the sequence of SEQ ID NO. 15, corresponding to the sequence of the ORF5 ectodomain in the sequence of SEQ ID NO. 1 encoding the porcine reproductive and respiratory syndrome virus strain CSNA11, is changed to the sequence of SEQ ID NO. 16 or 17. Claim 4 A polynucleotide represented by the base sequence of SEQ ID NO.

16. Claim 5 A polynucleotide represented by the base sequence of SEQ ID NO.

17. Claim 6 delete Claim 7 Polypeptide consisting of the amino acid sequence of SEQ ID NO.

18. Claim 8 Polypeptide consisting of the amino acid sequence of SEQ ID NO.

19. Claim 9 In claim 8, a polypeptide composed of the amino acid sequence of SEQ ID NO. 19 for increasing the production rate of neutralizing antibodies against porcine reproductive and respiratory syndrome virus. Claim 10 A method for producing an attenuated porcine reproductive and respiratory syndrome chimeric virus, characterized by including the step of inserting the vector of claim 2 into a cell. Claim 11 A method for producing an attenuated porcine reproductive and respiratory syndrome chimeric virus, characterized in that, in claim 10, the cell is MARC-145. Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 Attenuated porcine reproductive and respiratory syndrome chimeric virus produced by the manufacturing method of claim 10. Claim 16 Cells transformed with the vector of paragraph 2. Claim 17 A vaccine composition for the prevention or treatment of porcine reproductive and respiratory syndrome, characterized by comprising the attenuated porcine reproductive and respiratory syndrome chimeric virus of claim 3 or claim 15. Claim 18 A vaccine composition according to claim 17, characterized in that the vaccine composition further comprises an adjuvant.

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