RU2781446C1 - Chimeric porcine reproductive and respiratory syndrome virus and vaccine using said virus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely, to a chimeric porcine reproductive and respiratory syndrome (PRRS) virus. Proposed is a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) providing immunity against the PRRSV infection. The virus contains a polynucleotide consisting of a structural formula where the polynucleotide constitutes the chimeric virus genome: 5′-[X]-[Y]-3′, wherein [X] constitutes a gene fragment produced by processing the genome of the mutant strain LMY ver2 under the registration number 13394 BP with AscI and PacI restrictases; [Y] constitutes a gene fragment produced by processing the genome of the mutant strain BP2017-2 under the registration number 13393 BP with AscI and PacI restrictases. Proposed is a vaccine composition against the porcine reproductive and respiratory syndrome virus.

EFFECT: chimeric PRRSV according to the invention is weaker than the original strain thereof, therefore the virus stimulates the secretion of neutralising antibodies while having low pathogenicity and high stability, therefore being usable as a vaccine in the effective prevention and treatment of PRRS-associated diseases.

13 cl, 5 dwg, 12 tbl, 4 ex

Description

FIELD OF THE INVENTION

The present invention relates to a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) that can be used as a vaccine, and the chimeric virus of the present invention has low pathogenicity and high safety compared to its parent strain. The chimeric PRRSV virus of the present invention can enhance the secretion of neutralizing antibodies associated with cross-immunity and provide a vaccine that can provide effective protection against porcine reproductive and respiratory syndrome.

PRIOR ART

Porcine Reproductive Respiratory Syndrome (hereinafter referred to as PRRS) is an infectious disease that causes the greatest damage to pig production, along with porcine circovirus infection and foot-and-mouth disease. PRRS results in sterility in pregnant pigs, reproductive disorders such as miscarriage, premature birth or stillbirth, and respiratory symptoms such as sneezing, fever and the like in suckling pigs and finishing pigs. Generally, severe respiratory symptoms are caused by secondary bacterial infections or the like following infection with the virus, but in the case of chronic infection, there is a decrease in weight gain and an increase in mortality without characteristic clinical symptoms.

This viral disease was first discovered in 1987 in the United States of America, then in Europe and in the early 1990s in Asia. Until now, PRRS has been endemic in developed swine countries and has spread throughout the world, causing huge economic losses every year.

The causative agent of PRRS is the PRRS virus belonging to the genus Arterivirus, family Arteriviridae and order Nidovirales. The PRRS virus has a positive-sense single-stranded RNA genome and is approximately 15.4 kb in size. There are nine open reading frames (ORFs) in the PRRS virus genome (Conzelmann et al., 1993; Meulenberg et al, 1993). Of these, ORF1a and ORF1b encoding non-structural proteins (NSPs) comprise approximately 80% of the virus genome (Bautista et al, 2002; Meulenberg et al., 1993; Snijder and Meulenberg, 1998, 2001). Of these non-structural proteins, NSP1-alpha, NSP1-beta and NSP2-NSP8 are known to be located in ORF1a and NSP9-NSP12 in ORF1b. ORF2-7, making up the remaining 20%, encode the glycosylated structural proteins GP2, GP3, GP4, and GP5, the non-glycosylated membrane protein (M), and the nucleocapsid protein (N). The small structural proteins GP2, GP3, and GP4 form heterotrimers and act when the virus enters the host cell, while the large structural proteins GP5 and M form heterodimers and act to increase the infectivity of the virus.

The PRRS virus mutates heavily due to its RNA viral nature, so there are many differences between viruses. The PRRS virus is mainly divided into the North American type and the European type. There is type I (Lelystad virus, LV), which is a representative of the European type, and type II, which is a representative of the North American type of virus strain (North American strain) ATCC VR2332 (the genome sequence of VR2332 is listed in GenBank under accession number AY150564) (Murtaugh et al., Arch Virol 1995; 140:1451-1460).

It is known that the degree of difference between the genes of the North American type and the European type reaches 40%, so cross protection against both types is impossible. In addition, it is often not possible to obtain cross protection even against mutant strains belonging to the same type (Meng, X.J. et al., 2000). Therefore, a vaccine based on a standard mutant strain was made for each type, but this does not effectively prevent PRRS due to its insufficient ability to provide cross protection. To solve this problem, various attempts have been made to produce a vaccine that is effective in providing safety, immunogenicity and protection.

Due to the severity of the disease, in the approximately 20 years since the discovery of the porcine reproductive and respiratory syndrome virus (PRRSV), which is the causative agent of porcine reproductive and respiratory syndrome (PRRS), significant efforts have been invested in the development of a method for preventing infection with this virus, however methods for its prevention and treatment have not yet been developed. Various vaccines have been developed to control porcine reproductive and respiratory syndrome (PRRS), such as inactivated vaccines and attenuated live vaccines, but only attenuated vaccines with infectivity have been found to produce the desired level of protective effect. However, it is difficult to provide protection against different porcine reproductive and respiratory syndrome (PRRSV) viruses using a single vaccine due to the lack of cross-immunity to different types of viruses due to the wide variety of porcine reproductive and respiratory syndrome (PRRSV) viruses. In addition, existing attenuated vaccines can only be obtained by passage passage of 100-200 or more passages in cell lines of other animal species, resulting in a long development period and it is difficult to guarantee efficacy and safety. Therefore, a vaccine based on a standard mutant strain was made for each type, but this does not effectively prevent PRRS due to its insufficient ability to provide cross protection.

To solve this problem, various attempts have been made to produce a vaccine that effectively provides safety, immunogenicity and protection (application No. 10-2011-7004020; title of the invention: "Vaccination against highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS)").

DESCRIPTION OF THE INVENTION

Technical task

The present invention relates to a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) that has low pathogenicity and high safety and enhances the secretion of neutralizing antibodies, a vaccine containing said virus, and a method for producing the same.

In one example, the chimeric virus may comprise

(1) a nucleic acid sequence of open reading frame 1a (ORF1a) and ORF1b originating from the mutant strain LMY ver2 with accession number KSTS 13394 BP, or a nucleic acid sequence 70% or more homologous to said nucleic acid sequence while maintaining functional equivalence specified sequence, and

(2) the nucleic acid sequence of the ORF2-ORF7 regions of the isolated BP2017-2 strain having accession number KCTC 13393 BP, or a nucleic acid sequence that is 70% or more homologous to said nucleic acid sequence while maintaining functional equivalence to said sequence.

Technical solution

The present invention relates to a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) that can be used as a vaccine. The chimeric PRRSV virus of the present invention is attenuated and therefore has low pathogenicity and high safety compared to its parent strain, and enhances the secretion of neutralizing antibodies associated with cross-immunity, thereby greatly enhancing immunity in pigs. Accordingly, it can be used as an effective vaccine for the prevention and treatment of PRRS disease.

Hereinafter, the present invention will be described in more detail.

As used herein, "attenuated virus" refers to a non-toxic virus capable of inducing an immune response in a target mammal without resulting in clinical signs of PRRS disease, and also refers to a reduction in clinical signs in animals infected with or not receiving an attenuated virus, or a reduction in the severity of symptoms compared to "control" animals infected with unattenuated PRRS virus. In this context, the term "reduction/reduced" means a reduction of at least 10%, preferably 25%, more preferably 50%, or most preferably 100% or more compared to a control as defined above.

As used herein, a "vaccine composition" may be a chimeric PRRS virus, or any immunogenic fragment or region thereof, preferably an attenuated chimeric PRRS virus, such as the chimeric PRRS virus of the present invention. This results in an "immune response", such as a cellular and/or antibody-mediated immune response to PRRSV. Preferably, the vaccine composition is capable of conferring prophylactic immunity against PRRSV infection and associated clinical signs.

As used herein, "immune response" means any cell- and/or antibody-mediated immune response to a chimeric virus or vaccine when administered to an animal that is administered a chimeric PRRSV virus of the present invention or a vaccine composition containing said virus. Typically, an "immune response" includes, without limitation, one or more of the following: the appearance or activation of antigens that are part of the corresponding composition or vaccine, or antibodies, B cells, helper T cells, inhibitory T cells, and/or cytotoxic T cells, and/or 5 T cells specifically induced against antigens. Preferably, the host exhibits a therapeutic or prophylactic immune response that improves resistance to a new infection and/or reduces the clinical severity of the disease compared to a control not receiving the immunogenic composition or vaccine. This warning will be demonstrated, in the most favorable case, by the absence of symptoms associated with the above infection in the host, and, including this, by a decrease in frequency or severity.

When used here, "pigs", "pig" and "pigs" can be used in a compatible manner.

"Bringing into contact with the vaccine" or "administration of the vaccine" means the introduction of a chimeric PRRSV virus or a vaccine containing the specified chimeric PRRSV virus described here, prior to exposure leading to PRRS disease.

"Prevent" or "prevention" means a reduction in the incidence of clinical manifestations of PRRS or the severity or frequency of symptoms as a result of the administration of a PRRSV virus or a vaccine composition containing said PRRSV virus of the present invention. The reduction in severity or frequency is the result of comparison with an animal or group of animals without administration of a chimeric PRRSV virus or a vaccine composition containing said PRRSV virus of the present invention. Preferably, the animal may be a pig.

For convenience, the sequences indicated herein are described in terms of DNA nucleotides, and in the case of RNA-type polynucleotides, this means a sequence where all or part of the thymine (T) of the nucleotide sequence is replaced by uracil (U).

Here, "consisting of a sequence" in relation to a particular nucleotide sequence and/or amino acid includes all of containing the specified sequence, essentially containing the specified sequence and/or consisting of the specified sequence, and, if necessary, they can be appropriately replaced and used.

The present invention provides an attenuated chimeric porcine reproductive and respiratory syndrome virus (PRRSV) which is a viral disease affecting pigs.

To solve this problem, according to the present invention, a polynucleotide is proposed containing the structure of the structural formula 1:

[structural formula 1]

5'-[X]-[Y]-3'.

In this formula, [X] represents the nucleic acid sequence of the genes of the ORF1a and ORF1b regions of the mutant strain LMY ver2, having the registration number 13394 BP, or a nucleic acid sequence that is 70% or more homologous to the nucleic acid sequence of the genes of the ORF1a and ORF1b regions, containing the nucleic acid sequence of the NSP1 genes ( NSP1-alpha gene and NSP1-beta gene) of the mutant strain LMY ver2 having the accession number KSTC 13394 BP, or a nucleic acid sequence homologous by 70% or more in the range allowing to maintain a function equal to the function of the specified nucleic acid sequence, for example, containing

the nucleic acid sequence of the NSP1 genes of the mutant strain LMY ver2, or a nucleic acid sequence that is 70% or more homologous in a range that allows it to maintain a function equal to the function of the specified nucleic acid sequence, and [Y] is the nucleic acid sequence of the genes of the ORF2-ORF7 regions of the mutant strain BP2017-2 , having the registration number KSTS 13393 BP, or a nucleic acid sequence homologous by 70% or more in the range that allows you to maintain a function equal to the function of the specified nucleic acid sequence. As used herein, "equal function" may mean a function that is the same as the desired function, or qualitatively (activity) and/or quantitatively (level) similar to it. For example, the amino acid sequence of the protein encoded by the gene in question may be the same as the amino acid sequence of the protein encoded by the wild-type gene.

In one example, [X] may contain all kinds of point mutations (silent mutation) in the range allowing the amino acid sequence of the NSP1 protein encoded by the NSP1 gene to be conserved in the nucleic acid sequence of the NSP1 gene consisting of the nucleotide sequence of SEQ ID NO: 11, and, for example, it can be freely selected and used in a range aimed at reducing the CBP index of the NSP1 gene.

For example, [X] may be a mutation where one or more, 25 or more, 66 or more, 80 or more, or all of the 91 bases selected from the group consisting of position 222, position 225, position 237, position 240 are replaced , regulations 252, regulations 306, regulations 309, regulations 312, regulations 315, regulations 324, regulations 327, regulations 330, regulations 333, regulations 336, regulations 339, regulations 342, regulations 345, regulations 357, regulations 363, regulations 366, regulations 378, regulation 379, regulation 381, regulation 393, regulation 396, regulation 543, regulation 546, regulation 549, regulation 555, regulation 558, regulation 561, regulation 573, regulation 579, regulation 582, regulation 588, regulation 612, regulation 618, regulations 621, regulations 627, regulations 633, regulations 639, regulations 654, regulations 673, regulations 675, regulations 678, regulations 681, regulations 684, regulations 705, regulations 708, regulations 729, regulations 735, regulations 738, regulations 741, regulations 744 , provisions 747 , regulations 771, regulations 786, regulations 789, regulations 792, regulations 810, regulations 825, regulations 828, regulations 838, regulations 840, regulations 846, regulations 849, regulations 858, regulations 867, regulations 879, regulations 882, regulations 885, regulations 891, regulation 900, regulation 903, regulation 906, regulation 924, regulation 936, regulation 939, regulation 948, regulation 954, regulation 963, regulation 966, regulation 1026, regulation 1029, regulation 1038, regulation 1047, regulation 1053, regulation 1066, positions 1068, positions 1086, positions and positions 1110 of the nucleic acid sequence of the NSP1 gene, consisting of the nucleotide sequence of SEQ ID NO: 11.

In one example, [X] may contain, in the nucleic acid sequence of the NSP1 gene, consisting of the nucleotide sequence of SEQ ID NO: 11, one or more, 25 or more, 66 or more, 80 or more, or all of 91 mutations selected from the group, consisting of a mutation where G at position 222 is replaced by C, a mutation where C at position 225 is replaced by A, a mutation where T at position 327 is replaced by C, a mutation where A at position 240 is replaced by T, a mutation where T at position 252 is replaced by C, mutations where A at position 306 is replaced by C, mutations where T at position 309 is replaced by C, mutations where G at position 312 is replaced by A, mutations where C at position 315 is replaced by A , mutations where T at position 324 is replaced by C, mutations where C at position 327 is replaced by G, mutations where G at position 330 is replaced by A, mutations where C at position 333 is replaced by T, mutations where C is at position 336 is replaced by G, mutations where T at position 339 is replaced by C, mutations where A at position 342 is replaced by T, mutations where T in position 345 is replaced by A, mutations where A at position 357 is replaced by G, mutations where T at position 363 is replaced by A, mutations where T at position 366 is replaced by C, mutations where C at position 378 is replaced by T, mutations where C at position 379 is replaced by A, mutations where C at position 381 is replaced by G, mutations where T at position 393 is replaced by C, mutations where T at position 396 is replaced by A, mutations where T at position 543 is replaced by C, mutations where T at position 546 is replaced by C, mutations where C at position 549 is replaced by A, mutations where T at position 555 is replaced by C, mutations where T at position 558 is replaced by C, mutations where T at position 561 is replaced by A, mutations where C at position 573 is replaced by T, mutations where T at position 579 is replaced by C, mutations where G at position 582 is replaced by T, mutations where T at position 588 replaced by C, mutations where T at position 612 is replaced by C, mutations where G at position 618 is replaced by C, mutations where T at position 621 is replaced by C, mutations where A at position 627 replaced by T, mutations where T at position 633 is replaced by C, mutations where T at position 639 is replaced by G, mutations where C at position 654 is replaced by T, mutations where C at position 673 is replaced by T, mutations where C at position 675 is replaced by A, mutations where C at position 678 is replaced by T, mutations where C at position 681 is replaced by G, mutations where C at position 684 is replaced by G, mutations where G is at position 705 replaced by C, mutations where C at position 708 is replaced by T, mutations where A at position 729 is replaced by C, mutations where T at position 735 is replaced by C, mutations where G at position 738 is replaced by T, mutations, where T at position 741 is replaced by C, mutations where T at position 744 is replaced by C, mutations where T at position 747 is replaced by A, mutations where T at position 771 is replaced by C, mutations where T at position 786 is replaced to C, mutations where C at position 789 is replaced by T, mutations where T at position 792 is replaced by G, mutations where C at position 810 is replaced by T, mutations where T at position 825 is replaced n to C, mutations where C at position 828 is replaced by T, mutations where T at position 838 is replaced by C, mutations where G at position 840 is replaced by C, mutations where C at position 846 is replaced by G, mutations, where G at position 849 is replaced by A, mutations where A at position 858 is replaced by G, mutations where T at position 867 is replaced by C, mutations where T at position 879 is replaced by C, mutations where C at position 882 is replaced to G, mutations where C at position 885 is replaced by T, mutations where C at position 891 is replaced by T, mutations where T at position 900 is replaced by C, mutations where C at position 903 is replaced by A, mutations where T at position 906 is replaced by C, mutations where G at position 924 is replaced by T, mutations where T at position 936 is replaced by C, mutations where T at position 939 is replaced by C, mutations where A at position 948 is replaced by C, mutations where A at position 954 is replaced by C, mutations where A at position 963 is replaced by T, mutations where T at position 966 is replaced by C, mutations where A at position 1026 is replaced by G, m mutations where G at position 1029 is replaced by C, mutations where C at position 1038 is replaced by T, mutations where T at position 1047 is replaced by C, mutations where T at position 1053 is replaced by C, mutations where A at position 1066 is replaced by C, mutations where A at position 1068 is replaced by C, mutations where C at position 1086 is replaced by T, and mutations where T at position 1110 is replaced by C.

For example, [X] may be the nucleic acid sequence of the NSP1 gene (SEQ ID NO: 12) and/or NSPl-beta (SEQ ID NO: 1; a region of 609 5' bases of SEQ ID NO: 12) of the LMY ver2 mutant strain having accession number 13394 BP, or a nucleic acid sequence that is 70% or more homologous, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more in the range that allows you to maintain a function equal to the function of the specified nucleic acid sequence.

In addition, [Y] may be the nucleic acid sequence of the genes of the ORF2-ORF7 regions of the mutant strain BP2017-2 having the registration number KSTC 13393 BP, or a nucleic acid sequence homologous by 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more in the range to keep the function equal to the function specified nucleic acid sequence.

According to one example of the present invention, the presence of [A]n at the 3' end of the [Y] of structural formula 1 is also possible. Said n is the number of nucleotides containing base A and may be an integer from 10 to 100. may be an integer from 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 30, 20 to 30, 20 to 26.

The polynucleotide may be RNA, reverse transcriptome RNA (DNA), or a combination thereof. Said polynucleotide can function as the chimeric PRRSV virus genome.

Accordingly, according to another example of the present invention, there is provided a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) containing a polynucleotide having the structure of structural formula 1. The genome of the chimeric PRRSV virus may be DNA or RNA, preferably RNA.

[X] corresponds to the region of the NSP1 gene containing the NSP1-beta mutant strain LMY ver2 having accession number 13394 BP, and may be a gene fragment obtained by processing the genome of the mutant strain LMY ver2 with restriction enzymes AscI and PacI, and, for example, may contain a nucleic acid sequence SEQ ID NO: 1 and/or SEQ ID NO: 12, or nucleic acid sequence 70% or more homologous, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more homologous, 96 % or more, 97% or more, 98% or more, 99% or more, or 99.5% or more in the range that allows you to maintain a function equal to the function of the specified nucleotide sequence.

[Y] corresponds to the ORF2-ORF7 regions of the mutant strain BP2017-2, having the registration number KSTC 13393 BP, and may be a gene fragment obtained by processing the genome of the mutant strain BP2017-2 with restriction enzymes AscI and PacI, and, for example, may contain the nucleic acid sequence SEQ ID NO: 2 or nucleic acid sequence 70% or more homologous, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homologous, 98% or more, 99% or more, or 99.5% or more in the range that allows you to maintain a function equal to the function of the specified nucleic acid sequence.

In one example, structural formula 1 may contain a nucleic acid sequence of SEQ ID NO: 4 or a nucleic acid sequence that is 70% or more homologous, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more homologous , 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more in the range that allows you to maintain a function equal to the function of the specified nucleic acid sequence. For example, it may be the nucleic acid sequence of SEQ ID NO: 4.

In Table 1 below, the bold portion of the nucleotide sequence of the NSP1 gene, LMY ver2 is the NSP1 beta region.

(In the following table, a polynucleotide (RNA) provided herein may contain a nucleic acid sequence where, in the sequence shown in SEQ ID NOs: 1-4, T has been replaced by U.)

The present invention provides a chimeric PRRSV virus containing a polynucleotide of structural formula 1 as a genome. The chimeric PRRSV virus may include viral progeny cultured at passages 1-80, passages 1-70, passages 1-60, passages 1-50, passages 1-40, passages 1-30, passages 1-20, or passages 1-10 .

[structural formula 1]

5'-[X]-[Y]-3'.

In the above formula, [X] represents the nucleic acid sequence of the NSP1 gene and/or NSP1 beta mutant strain LMY ver2 having accession number 13394 BP, or a nucleic acid sequence 70% or more homologous to the specified nucleic acid sequence, and [Y] represents the nucleic acid the gene sequence of the ORF2-ORF7 regions of the BP2017-2 mutant strain having the registration number KCTC 13393 BP, or a nucleic acid sequence that is 70% or more homologous to the specified nucleic acid sequence.

For example, [X] may be the nucleic acid sequence of the NSP1 gene and/or NSP1 beta mutant strain LMY ver2 having accession number 13394 BP, or the nucleic acid sequence of 70% or more, 75% or more, 80% or more, 85 % or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homologous to the specified nucleic acid sequence.

In addition, [Y] may be the nucleic acid sequence of the genes of the ORF2-ORF7 regions of the mutant strain BP2017-2 having the accession number KSTC 13393 BP, or the nucleic acid sequence of 70% or more, 75% or more, 80% or more, 85 % or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homologous to the specified nucleic acid sequence.

According to one example of the present invention, the presence of [A]n at the 3' end of the [Y] of structural formula 1 is also possible. Said n is the number of nucleotides containing base A and may be an integer from 10 to 100, and for example , can be an integer from 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 15 to 80, 15 to 70, 15 to 60 , 15 to 50, 15 to 40, 15 to 30, 20 to 30, 20 to 26.

The polynucleotide may be RNA, reverse transcriptome RNA (DNA), or a combination thereof. Said polynucleotide can function as the chimeric PRRSV virus genome. Accordingly, according to another example of the present invention, there is provided a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) containing a polynucleotide having the structure of structural formula 1. The genome of the chimeric PRRSV virus may be DNA or RNA, preferably RNA.

[X] corresponds to the region of the NSP1 gene containing the NSP1-beta mutant strain LMY ver2 having accession number 13394 BP, and may be a gene fragment obtained by processing the genome of the mutant strain LMY ver2 with restriction enzymes AscI and PacI, and, for example, may contain a nucleic acid sequence SEQ ID NO: 1 or nucleic acid sequence 70% or more homologous, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homologous , 98% or more, 99% or more, or 99.5% or more in the range that allows you to keep the function equal to the function of the specified nucleotide sequence.

[Y] corresponds to the ORF2-ORF7 regions of the mutant strain BP2017-2, having the registration number KSTC 13393 BP, and may be a gene fragment obtained by processing the genome of the mutant strain BP2017-2 with restriction enzymes AscI and PacI, and, for example, may contain the nucleic acid sequence SEQ ID NO:2 or nucleic acid sequence 70% or more homologous, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homologous, 98% or more, 99% or more, or 99.5% or more in the range that allows you to maintain a function equal to the function of the specified nucleic acid sequence.

In one example, structural formula 1 may contain a nucleic acid sequence of SEQ ID NO: 4 or a nucleic acid sequence that is 70% or more homologous, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more homologous , 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more in the range that allows you to maintain a function equal to the function of the specified nucleic acid sequence. For example, it may be the nucleic acid sequence of SEQ IN NO: 4.

According to one specific example, a chimeric PRRSV virus is provided, containing a nucleic acid sequence containing a polynucleotide of SEQ ID NO: 4, as a genome, named LMY+BP2017. The LMY+BP2017 virus may be a virus with accession number KCTC 13394 BP.

The mutant strain LMY ver2, which is the backbone of the chimeric virus LMY+BP2017, may be a mutant strain PRRSV containing a mutant NSP1-beta, where at least one base of the non-structural protein type 1 (NSP1) SEQ ID NO: 5 of the original LMY strain is replaced (registration number in GenBank DQ473474.1.).

The nucleotide sequence to be replaced in the nucleotide sequence of the gene encoding NSP1 and/or NSPl-beta strain LMY can be selected using the well-known SAVE (Synthetic Attenuated Virus Engineering) program, and preferably can be selected using the SAVE program developed by the authors of the present invention. Specifically, it may be a de-optimized nucleotide sequence obtained by analyzing the genetically highly stable region of NSP1 in the genome of the original LMY strain using the SAVE program developed by the authors of the present invention, followed by selection and replacement of part or all of the nucleotide sequence showing a relatively high preference for codon pairs (Codon Pair Bias, SRV). The deoptimized nucleotide sequence may contain, in the gene encoding the NSP1 protein of the LMY strain, consisting of the nucleotide sequence of SEQ ID NO: 11, one or more, 25 or more, 66 or more, 80 or more, or all of the 91 mutations selected from the group, consisting of a mutation where G at position 222 is replaced by C, a mutation where C at position 225 is replaced by A, a mutation where T at position 327 is replaced by C, a mutation where A at position 240 is replaced by T, a mutation where T at position 252 is replaced by C, mutations where A at position 306 is replaced by C, mutations where T at position 309 is replaced by C, mutations where G at position 312 is replaced by A, mutations where C at position 315 is replaced by A , mutations where T at position 324 is replaced by C, mutations where C at position 327 is replaced by G, mutations where G at position 330 is replaced by A, mutations where C at position 333 is replaced by T, mutations where C is at position 336 is replaced by G, mutations where T at position 339 is replaced by C, mutations where A at position 342 is replaced by T, mutations where T at position 345 is replaced by A, mutations where A at position 357 is replaced by G, mutations where T at position 363 is replaced by A, mutations where T at position 366 is replaced by C, mutations where C at position 378 is replaced by T, mutations where C at position 379 is replaced by A, mutations where C at position 381 is replaced by G, mutations where T at position 393 is replaced by C, mutations where T at position 396 is replaced by A , mutations where T at position 543 is replaced by C, mutations where T at position 546 is replaced by C, mutations where C at position 549 is replaced by A, mutations where T at position 555 is replaced by C, mutations where T in position 558 is replaced by C, mutations where T at position 561 is replaced by A, mutations where C at position 573 is replaced by T, mutations where T at position 579 is replaced by C, mutations where G at position 582 is replaced by T, mutations where T at position 588 is replaced by C, mutations where T at position 612 is replaced by C, mutations where G at position 618 is replaced by C, mutations where T at position 621 is replaced by C, mutations and where A at position 627 is replaced by T, mutations where T at position 633 is replaced by C, mutations where T at position 639 is replaced by G, mutations where C at position 654 is replaced by T, mutations where C at position 673 is replaced by T, mutations where C at position 675 is replaced by A, mutations where C at position 678 is replaced by T, mutations where C at position 681 is replaced by G, mutations where C at position 684 is replaced by G, mutations where G at position 705 is replaced by C, mutations where C at position 708 is replaced by T, mutations where A at position 729 is replaced by C, mutations where T at position 735 is replaced by C, mutations where G at position 738 replaced by T, mutations where T at position 741 is replaced by C, mutations where T at position 744 is replaced by C, mutations where T at position 747 is replaced by A, mutations where T at position 771 is replaced by C, mutations, where T at position 786 is replaced by C, mutations where C at position 789 is replaced by T, mutations where T at position 792 is replaced by G, mutations where C at position 810 is replaced by T, mutations where T is at position 825 is replaced by C, mutations where C at position 828 is replaced by T, mutations where T at position 838 is replaced by C, mutations where G at position 840 is replaced by C, mutations where C at position 846 is replaced by G, mutations where G at position 849 is replaced by A, mutations where A at position 858 is replaced by G, mutations where T at position 867 is replaced by C, mutations where T at position 879 is replaced by C, mutations where C at position 882 replaced by G, mutations where C at position 885 is replaced by T, mutations where C at position 891 is replaced by T, mutations where T at position 900 is replaced by C, mutations where C at position 903 is replaced by A, mutations where T at position 906 is replaced by C, mutations where G at position 924 is replaced by T, mutations where T at position 936 is replaced by C, mutations where T at position 939 is replaced by C, mutations where A is at position 948 replaced by C, mutations where A at position 954 is replaced by C, mutations where A at position 963 is replaced by T, mutations where T at position 966 is replaced by C, mutations where A at position 1026 replaced by G, mutations where G at position 1029 is replaced by C, mutations where C at position 1038 is replaced by T, mutations where T at position 1047 is replaced by C, mutations where T at position 1053 is replaced by C, mutations , where A at position 1066 is replaced by C, mutations where A at position 1068 is replaced by C, mutations where C at position 1086 is replaced by T, and mutations where T at position 1110 is replaced by C, without limitation to these options.

Using the SAVE program, it is possible to quantify the CPV, which is a preference formed during the formation of codon pairs in viral genes, using a computer algorithm, and since the proliferative properties of the virus decrease with a decrease in the CPV index (deoptimization) and its weakening (Virus Attenuation by Genome -Scale Changes in Codon Pair Bias, Science, 2008, J. Robert Coleman et al), the attenuated LMY ver2 mutant strain used to produce the chimeric virus of the present invention can be obtained by replacing a portion of the nucleotide sequence with a high CRP score with a nucleotide sequence with a low CPV or, preferably, by conducting silent mutations in accordance with the principle of deoptimization of codon pairs. The LMY ver2 mutant strain may contain a gene encoding an NSP1-beta protein consisting of the nucleotide sequence of SEQ ID NO:6 or a gene encoding an NSP1 protein consisting of the nucleotide sequence of SEQ ID NO:12.

In addition, according to the present invention, a cell containing the genome of the chimeric PRRSV virus of the present invention can be provided. A cell refers to a cell transfected with a chimeric virus genome (DNA, RNA, or a vector containing the specified DNA or RNA) or a chimeric virus containing the specified genome to obtain a large amount of the chimeric virus, and the cell type is not limited as long as it is suitable for the task.

According to one example of the present invention, a porcine reproductive and respiratory syndrome virus vaccine composition comprising a chimeric PRRSV virus or subcultured progeny thereof can be provided.

Subcultured progeny may include viral progeny subcultured at passages 1-80, passages 1-70, passages 1-60, passages 1-50, passages 1-40, passages 1-30, passages 1-20, or passages 1-10.

According to one example of the present invention, the vaccine may be a live vaccine or an inactivated vaccine, but preferably it is a live vaccine. Specifically, the attenuated chimeric PRRS virus described herein may be a modified live vaccine containing one or more of the above viral strains in a viable state in a pharmaceutically acceptable carrier. Moreover, or alternatively, an inactivated virus can be used to make an inactivated vaccine.

The vaccine may further contain one or more selected from the group consisting of carriers, diluents, excipients and adjuvants. The type of pharmaceutically acceptable carrier is not limited, but may include any and all solvents, dispersion media, coatings, stabilizers, preservatives, antibiotics and antifungal agents, isotonic agents, absorption delaying agents, and the like.

On the other hand, an effective dose of an attenuated chimeric virus of the present invention present in a vaccine composition may be that amount of virus that results or can result in an immune response in an animal administered with said effective dose of virus. The effective amount may depend on the components of the vaccine and the administration schedule. The dose of the vaccine composition may range from 2 to 6, preferably 3 to 4 cytopathic doses of 50% (TCID ₅₀ ), and may vary depending on the type of subject, without being limited to these options.

The genome of the chimeric LMY-BP2017 virus of the present invention and the vaccine composition containing said genome can be used to prevent the effects of PRRS disease in pigs. In addition, subunits containing immunogenic fragments or regions of a chimeric PRRS virus can be used to prevent the effects of PRRS disease in pigs. The attenuated chimeric virus of the present invention, or a vaccine composition containing said virus, may be administered prophylactically prior to exposure of a pig to a strain of PRRS virus that causes PRRS, and may be administered to a pig simultaneously with exposure of a pig to such a virus strain, and may be administered therapeutically after exposure. the pig in question with that virus strain.

The vaccine composition of the present invention can be used to prevent porcine reproductive and respiratory syndrome (PRRS), for example, to prevent PRRS caused by the North American type of PRRSV. In one example, the North American type of PRRSV may be the type II virus strain VR2332.

The attenuated chimeric PRRSV virus of the present invention or a vaccine composition containing said virus can be administered by oral, parenteral, subcutaneous, intramuscular, intradermal, sublingual, transdermal, rectal, mucosal, inhalation, based on surface area, buccal administration or combinations thereof. In addition, the attenuated chimeric PRRS virus may be administered in the form of an implant capable of sustained release of the attenuated virus.

The attenuated chimeric PRRSV virus of the present invention or a vaccine composition containing said virus can be administered by injection, inhalation or transplantation, and injection is particularly preferred. Depending on the desired period and the effectiveness of vaccination or treatment, the attenuated chimeric PRRSV virus or a vaccine composition containing said virus can be administered one or more times and periodically, for example, over several days, several weeks or several months, at different doses each. day. Injection administration can be carried out by injection in the desired amount, or by subcutaneous injection, or by spraying into the nasal cavity, or, alternatively, by continuous injection.

According to one example of the present invention, the chimeric PRRSV virus of the present invention may be a virus having a TCID ₅₀ value of 0.01 to 0.1 compared to the TCID ₅₀ value of the parent strain, which is the TCID ₅₀ value of the virus measured bringing porcine alveolar macrophages into contact with the chimeric virus and its parent strain (GenBank accession number DQ473474.1.). Preferably, the measurement is taken after 2 days and the TCID ₅₀ value may be between 0.05 and 0.1. The chimeric PRRSV virus of the present invention has a TCID ₅₀ less than the parent strain, less proliferative properties, and is attenuated.

In addition, a virus is provided, wherein the mutant strain has a neutralizing antibody content, measured by introducing a chimeric PRRSV virus of the present invention and its parent strain (GenBank accession number DQ473474.1.) to a pig, from 2 to 8 relative to the parent strain, preferably from 2 up to 4. The measurement is preferably carried out after 28 days. Neutralizing antibody means an antibody having a neutralizing ability against VR2332, a representative strain of the North American type of PRRSV. Accordingly, when an attenuated chimeric virus of the present invention or a vaccine composition containing said virus is administered to a pig, the pig will experience an increase in neutralizing antibody titer and expression of immune factors, so that the effect of immunization against PRRS can be significantly increased.

According to one specific example, the CPV value of the chimeric PRRSV virus LMY+BP2017 of the present invention described above is less than that of the parent LMY strain (GenBank accession number DQ473474.1.), and may preferably be from -0.39 to 0 , more preferably -0.35 to -0.20, suitably -0.35 to -0.26, for example -0.2393184052058128. When the CPV is less than -0.39, it is difficult to obtain a virus, and when it is greater than 0, the problem is that it can be obtained, but the proliferative properties will not be reduced and thus it will not be attenuated. Accordingly, it is preferable that the attenuated LMY+BP2017 chimeric virus of the present invention has a CPB value as indicated above.

In addition, according to the present invention, a chimeric LMY+BP2017 virus is provided, having CpG, Up A values ranging from 1.0 to 3.0 relative to the original strain (GenBank registration number DQ473474.L). A change in the ratio of CpG, UpA is a necessary result of deoptimization, and an increase in the CpG, UpA of eukaryotic genes leads to cellular stress, and therefore there is a decrease in the proliferative properties of the virus and, thus, weakening of the virus is possible. The LMY+BP2017 chimeric virus of the present invention has higher CpG, UpA than the parent strain, and preferably 1.0 to 3.0 times greater, and for example, the attenuated chimeric virus of the present invention may have CpG 1.1753 and UpA 1.03.

According to one example of the present invention, a method for obtaining a chimeric porcine reproductive and respiratory syndrome virus is proposed, including processing the genome of the mutant strain LMY ver2, having the registration number 13394 BP, with AscI and PacI restrictases to obtain a polynucleotide fragment, processing the genome of the mutant strain BP2017-2, having the registration number KSTC number 13393 BP, restriction enzymes AscI and PacI to obtain a polynucleotide fragment and recombination of the LMY ver2 polynucleotide fragment and the BP2017-2 polynucleotide fragment to obtain an infectious clone.

The polynucleotide fragment obtained by processing the genome of the LMY ver2 mutant strain with restriction enzymes AscI and PacI may contain a region encoding NSP1-beta.

The polynucleotide fragment obtained by processing the genome of the mutant strain BP2017-2 with restriction enzymes AscI and PacI may contain ORF2-ORF7 regions.

When recombining the fragments, a ligase can be used, and a chimeric virus can be obtained by introducing the resulting infectious clone into a cell.

Cell refers to a cell transfected with a DNA or RNA of a chimeric virus or a vector containing said DNA or RNA, an infectious clone or a chimeric virus to obtain a large amount of the chimeric virus, and the cell type is not limited.

In addition, according to one example of the present invention, a composition for the prevention or treatment of porcine reproductive and respiratory syndrome virus containing the specified vaccine composition can be provided.

In a preferred form, the composition for the prevention or treatment of porcine reproductive and respiratory syndrome virus of the present invention may contain an additional component known to those skilled in the art, and may additionally contain suitable carriers, excipients and diluents commonly used for the manufacture of a pharmaceutical composition.

Moreover, it can be used as an oral administration form such as powders, granules, tablets, capsules, suspension, emulsion, syrup, aerosols and the like, an external preparation, a suppository and a sterile injection. Preferably, the use of a suitable composition known in the art, as disclosed in the relevant document (Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA).

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In making the composition, it may be made using a diluent or excipient such as a filler, diluent, binder, wetting agent, disintegrant, surfactant, and the like. Solid compositions for oral administration include tablets, pills, powder, granules, capsules, and the like, and these solid compositions are made by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like, with the composition. In addition, in addition to a simple excipient, a lubricating agent such as magnesium stearate and talc. Liquid compositions for oral administration include suspension, oral liquid, emulsion, syrup, and the like, and, in addition to commonly used simple diluents, water, and liquid paraffin, may contain various excipients, for example, wetting agent, sweetener, flavoring agent, preservative. etc. Compositions for parenteral administration include sterile aqueous solution, non-aqueous solvents, suspension, emulsion, lyophilized compositions and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, an injectable ester such as ethyl oleate, and the like can be used. Witepsol, Macrogol, Tween 61, cocoa butter, laurin (laurinum), glycerogelatin and the like can be used as the basis of suppositories.

The preferred dosage of the composition of the present invention varies depending on the condition and body weight of the subject, the severity of the disease, the dosage form, and the route and period of administration, but may be suitably selected by those skilled in the art. For example, for a preferred effect, the composition of the present invention can be administered in an amount of 0.0001 to 1000 mg/kg (body weight) per day. The introduction of the composition can be carried out once a day or divided into several times.

The composition of the present invention may be administered to a subject in a variety of ways. Any route of administration may be contemplated.

According to another example of the present invention, a method for preventing or treating porcine reproductive and respiratory syndrome can be provided, comprising administering to a pig a vaccine composition of the present invention.

Administration of a vaccine composition containing the genome of the chimeric virus of the present invention to a pig can enhance the pig's immune response to PRRSV antigens, and by enhancing the pig's immune response to PRRSV antigens, a method for preventing or treating porcine reproductive and respiratory syndrome can be provided. Preferably, it may occur a warning.

Specifically, the method may include administering the composition by subcutaneous injection, intravenous injection, intradermal injection, parenteral injection, intramuscular injection, needleless injection, electroporation, oral delivery, intranasal delivery, oronasal delivery, or any combination thereof.

According to the present invention, a kit may be provided for carrying out any of the above methods. This kit may contain a container, a vaccine composition preferably containing an attenuated chimeric PRRS virus of the present invention, a pharmaceutically acceptable carrier, an adjuvant, and instructions for administering the immunogenic composition to an animal in need thereof to reduce the clinical signs or effects of PRRS infection, preferably the frequency or severity PRRS. The kit may also contain means for injection and/or other types of means for administration. In addition, the kit may contain a solvent. The attenuated vaccine may be lyophilized and reconstituted with a solvent to form a solution for injection and/or inhalation. The solvent may be water, saline, buffer, or enhancement solvent. The kit may contain separate containers containing the attenuated virus, diluent and/or pharmaceutically acceptable carrier. The instructions may be labels and/or printed instructions attached to one or more containers.

Useful effects

The chimeric PRRSV virus of the present invention can be used to effectively prevent PRRS by substantially enhancing the secretion of neutralizing antibodies when administered to pigs, and can be usefully used as a vaccine for the treatment of PRRS.

BRIEF DESCRIPTION OF GRAPHICS

FIG. 1 shows the general genomic structure of the PRRS virus.

FIG. 2 is a schematic diagram showing the genomic structure of a chimeric LMY+BP2017 virus synthesized by excising part of a clone of the LMY ver2 virus and the BP2017-2 virus, respectively, and ligating them.

FIG. 3 shows the phylogenetic tree of the PRRSV virus LMY ver2 and the chimeric virus LMY+BP2017.

FIG. 4 shows the differences in the proliferative properties of PRRSV virus LMY ver2 and chimeric virus LMY+BP2017 in porcine alveolar macrophages (PAM cells).

FIG. Figure 5 compares differences in neutralizing antibody secretion after administration of PRRSV LMY, LMY+BP2017 chimeric virus, and phosphate buffered saline (PBS) to pigs.

EMBODIMENT OF THE INVENTION

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are provided only to explain the present invention, and the present invention is not limited to the following examples.

Since RNA is easily degraded, after all the actions were carried out by converting it into DNA, the cells were transfected, synthesizing RNA from the resulting DNA.

Example 1: Obtaining a chimeric virus 1-1.

Construction of a chimeric virus

A chimeric PRRS virus was constructed containing a non-structural protein region (non-structural protein type 1, NSP1; SEQ ID NO: 11) of a mutant strain of strain LMY and regions of ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7 of BP2017-2 isolated in 2017 G.

1-2. Obtaining an attenuated mutant strain of LMY

The LMY ver2 mutant strain was obtained by replacing the 91 bases of the nucleotide sequence of the NSP1 region gene using the LMY strain (GenBank accession number DQ473474.1.), which is a PRRS strain isolated by the traditional Quarantine Agency. In the NSP1 region gene, 25 bases of the NSP1-alpha region and 66 bases of the NSP1-beta region were replaced.

Specifically, the recombinant LMY ver2 strain was obtained by silently mutating a portion of the nucleotide sequence according to the principle of codon pair deoptimization (Table 3) using the well-known SAVE (Synthetic Attenuated Virus Engineering) program or the SAVE (Synthetic Attenuated Virus Engineering) program. ) developed by the authors of the present invention. First, the SAVE program was used to quantify the CPW (Codon Pair Preference), which is a preference formed by the codon interaction of the LMY gene when they are paired, using a computer algorithm. The CRV index changes when some nucleotide sequences of the LMY virus genes are replaced with other nucleotide sequences and is closely related to the proliferative properties of the virus. The proliferative properties of the virus are reduced and weakened by deoptimization of the CPB index through substitutions in the nucleotide sequence (Virus Attenuation by Genome-Scale Changes in Codon Pair Bias, Science, 2008, J. Robert Coleman et al.). The present inventors selected an NSP1 region (NSP1-alpha and NSP1-beta, SEQ ID NO: 11) with high genetic stability in the genome of the original LMY strain, analyzed it with the SAVE program, and selected 25 bases in the NSP1-alpha region and 66 bases in the NSP1- beta, a total of 91 bases, from base regions with relatively high CPB values to obtain a mutant strain of the LMY virus. A mutant strain of LMY virus with a 91 nucleotide mutation in the sequence of NSP1 (SEQ ID NO: 12) obtained by the described method was named LMY ver2, and its nucleotide sequence is shown in Table 3 below. LMY ver2 has registration number 13394 BP.

(In Table 3, regions of the NSP1 LMY ver2 gene sequences highlighted in bold underlined characters represent regions with base mutations in the NSP1 LMY gene or the NSP1-beta LMY gene.)

Then, to confirm the degree of attenuation of the LMY ver2 mutant strain, the CRP was measured. The measured CPV of NSP1 of the original LMY strain was approximately 0.0139 and the measured CPB of NSP1-beta was approximately 0.016, but the measured CPB of the LMY ver2 mutant strain of the present invention was approximately -0.2393 for NSP1 and approximately -0.33 for NSP1-beta. Based on this, it was concluded that the mutant strain LMY ver2 of the present invention decreased in proliferative properties compared to the traditional parent strain and that this strain is attenuated.

Then received infectious clone LMY ver2.

First, the full-length gene sequence of the LMY strain (registration number in GenBank DQ473474.1.) was divided into 7 fragments and synthesized, respectively. Of these 7 fragments, fragment 1 was the NSP1 region, and this region was synthesized as a DNA fragment (SEQ ID NO: 12) with a 91 base substitution in the gene nucleotide sequence of the NSP1 region of LMY strain. The synthesized gene fragment was sequentially cut with the restriction enzymes shown in Table 5 below and ligated with ligase to obtain one infectious clone.

1-3. Construction of a chimeric viral clone

According to the schematic representation in FIG. 1, the PRRS virus contains a total of 8 ORFs, namely ORF1a, ORF1b and ORF2-7.

From the genomic region containing the full-length structural gene of the LMY mutant strain ver2 amplified in Example 1-1, the ORF2-ORF7 regions were excised using AscI and PacI restriction enzymes. Then, the genomic region of the BP2017 strain, that region corresponding to the regions of ORF2-ORF7 of the mutant strain LMY ver2, was cut with the same restrictases AscI and PacI, and then the region of ORF1a and ORF1b of the mutant strain LMY ver2 and the region corresponding to the regions of ORF2-ORF7 of the strain BP2017 , were ligated with ligase to obtain a recombinant infectious clone named LMY+BP2017. The restriction enzymes used are shown in Table 6 below.

The finished infectious clone was introduced into a high copy number vector into which the CMV promoter and ampicillin resistance gene were introduced, and then transfected with BHK (Korean Cell Line Bank) cells using lipofectamine, and as a result, the chimeric LMY+BP2017 virus of the present invention was obtained.

The LMY+BP2017 chimeric virus was deposited at the Microbial Resource Center of Korea Research Institute of Bioscience and Biotechnology on October 24, 2018 with registration number KCTC13675 BP.

Example 2 Measurement of CpG and UpA of LMY+BP2017 chimeric virus

To assess the proliferative properties of the LMY+BP2017 chimeric virus obtained in Example 1, the CpG and UpA ratios of the NSP1 gene of the LMY+BP2017 chimeric virus and the CpG and UpA values of the NSP1-beta gene of the original LMY strain were measured.

A change in the CpG and UpA ratios of the viral gene is an inevitable result of deoptimization, and an increase in the CpG and UpA values of this gene leads to cellular stress and a decrease in the proliferative properties of the virus. The ratios of CpG and UpA of the NSP1-beta gene of the LMY+BP2017 chimeric virus and the LMY virus were measured using the SSE program (version 1.2) and the results are shown in Table 7 below.

As shown in Table 7, it was confirmed that the CpG and UpA of the LMY+BP2017 chimeric virus were increased compared to the original LMY strain, and based on this, it was possible to confirm a significant decrease in the proliferative properties of the LMY+BP2017 chimeric virus compared to the original LMY strain. .

Example 3. Identification of the chimeric strain LMY+BP2017 3-1. Identification of the chimeric strain LMY+BP2017

To identify the chimeric strain LMY+BP2017, the NSP1 regions of the chimeric strain LMY+BP2017 synthesized in Example 1 and the LMY strain (GenBank accession number DQ473474.1.) were sequenced.

Specifically, PCR was performed using the primer set shown in Table 8, allowing detection of 1-1654 nucleotides, including the NSP1 region, of the LMY virus and a one-step RT-PCR kit (Intron) and, specifically, after an initial RT at 45°C for 30 minutes and 95°C for 5 minutes, the reaction was carried out at 94°C for 30 seconds, at 61°C for 30 seconds and at 72°C for 2 minutes, repeating this cycle 38 times. The PCR amplified products were then sequenced, completing the identification of each strain.

3-2. Comparison of homology with other strains and phylogenetic tree

To confirm genetic differences between the recombinant LMYver2 virus strain of the present invention obtained in Example 1 and the known PRRS virus strain, the sequence identity of each strain in the NSP1 region was measured using the Bio Edit program, and the homology comparison results are shown in Table 9.

As shown in Table 9, it can be seen from the results of the homology comparison that the LMY+BP chimeric virus has a unique sequence different from other PRRS strains.

Based on the homology comparison results, a phylogenetic tree was analyzed using the BioEdit program, repeating the analysis ("Bootstrap") 1000 times. Based on this, an approximate phylogenetic tree of the chimeric virus LMY+BP2017 and LMY was constructed, shown in FIG. 3. Based on the phylogenetic tree shown in FIG. 3, the systematic position of the present virus can be determined.

3-3. Cell passage study

The persistence of the modified region during passage of the LMY+BP2017 chimeric virus obtained in Example 1 was confirmed. to confirm gene mutations, sequencing was performed every 10 passages using the same method as in Example 3-1.

As a result of confirmatory sequencing, it was confirmed that the region of substitutions in the gene of the present virus remained unchanged even after stabilization by 30 passages. Table 10 below shows the number of base mutations (nucleotide changes), mutation sites, and amino acid mutations (amino acid changes) after each number of passages.

As confirmed in Table 10, even after more than 30 passages, the NSP1a or NSP1b ORF1a gene mutations remained unchanged and only NSP2 or ORF2a protein mutations were detected. Thus, the persistence of mutations in the gene encoding the NSP1 protein in the virus obtained in Example 1 was confirmed even after 40 passages.

Example 4: Comparison of proliferative properties of LMY and chimeric LMY+BP

To confirm the proliferative properties of the LMY+BP2017 chimeric virus obtained in Example 1, the amount of virus obtained after bringing macrophages (PAM, Porcine Alveolar Macrophage [porcine alveolar macrophage]) into contact with the LMY+BP2017 chimeric virus and its parental strain was confirmed by measuring TCID ₅₀ .

Specifically, PAM cells were aliquoted at 2×10 ⁶ cells per well in a 6-well plate containing 2 ml of RPMI medium (containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin). LMY and LMY+BP2017 chimeric virus were then added to different wells, respectively, at an MOI ratio of 0.01, 1 hour after the addition, the entire supernatant was removed and aliquoted with 2 ml of RPMI maintenance medium solution (containing 10% FBS, 1% penicillin and streptomycin ). Two days after aliquoting, the supernatant was collected from each well and the TCID ₅₀ was measured. For the cells used in the subsequent experiment, MARC-145 cells were adsorbed to the wells of a 96-well plate at 2×10 ⁵ cells per well by aliquoting with 100 μl of Dulbecco's Modified Eagle's Medium (DMEM) (10% (v/v .) FBS, 1% (w/v) penicillin, streptomycin) in advance, the day before the TCID ₅₀ measurement.

The introduced virus was prepared as follows. The leftmost well of a row of a 96-well plate was aliquoted with 200 μl of the pre-collected supernatant, and 180 μl of DMEM (FBS without antibiotics) was aliquoted into the other wells. Then, using a multipipette, 20 µl were taken from each well, starting from the left, and a tenfold dilution was carried out, aliquoting them into the well on the right. The dilution was carried out sequentially, changing the tips, and the last 12th well was left as a negative control. The obtained diluted solution of the virus was added to the plate, on which MARC-145 cells were adsorbed. Then the entire culture solution of MARC-145 cells was removed, 200 μl of PBS was aliquoted into each well and removed by repeating washing 3 times. After that, the resulting virus solution was added, aliquoting 100 µl into each well, and 2 hours after the introduction, the solution was removed and aliquoted with 100 µl of a new maintenance solution (DMEM medium, 10% FBS, 1% penicillin, streptomycin).

For approximately 7 days after aliquoting, aggregation and apoptosis, which are varieties of CPE (cytopathogenic effect), were confirmed in cells, and finally, the dilution factor at which CPE was present in 4 wells, representing half of the 8 solutions in which virus based on the dilution factor just before the well without CPE. The final value was calculated by multiplying by 10, based on 1 ml.

The LMY+BP2017 chimeric strain and the original LMY strain were contacted with porcine alveolar macrophages (PAM cells), and TCID ₅₀ values measured over 1-2 days are shown in Table 11 below and in FIG. four.

As can be seen from Table 11 and FIG. 4, according to the TCID ₅₀ measured on the 2nd day of strain introduction, when the virus begins to form in large quantities, the chimeric LMY+BP2017 showed a 100-fold lower TCID ₅₀ than LMY. Based on this, it could be seen that the proliferative properties of the LMY+BP2017 chimeric virus of the present invention in PAM cells (porcine alveolar macrophages), which are the main infected cells in PRRS, were at least 10 times less than that of LMY, and it was possible to confirm that the chimeric LMY+BP2017 virus of the present invention was attenuated because its proliferative properties were less than that of the original LMY strain.

Example 4: Confirmation of the Secretion Pattern of Neutralizing Antibodies

To confirm the secretion pattern of neutralizing antibodies after administration of the chimeric virus of the present invention, 12 pigs at 3 weeks of age were divided into 3 groups of four pigs each and injected with LMY (10 ⁵ TCID ₅₀ ), chimeric virus LMY+BP2017 (10 ⁵ TCID ₅₀ ) and PBS, 2 ml each. After 4 weeks, venous blood was obtained from pigs from each group, 2 ml of serum was isolated, and the amount of neutralizing antibodies in the serum was measured.

Specifically, the analysis was performed by a publicly known method. Prior to analysis, all serum samples were inactivated by heating to 56°C for 45 minutes. Inactivated serum was added to RPMI 1640 medium (10% FCS, 20 mM L-glutamine, a mixture of antibiotics and antifungal agents: 100 IU/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, 0.25 µg/ml amphotericin B ) and then a two-fold dilution was performed. After that, 100 μl of each diluted serum was taken and mixed with 100 μl of the previously prepared VR2332 strain, 200 TCID ₅₀ /ml, incubated at 37°C for 1 hour and then brought into contact with a monolayer of Marc-145 cells cultured the day before. this. Then, after incubation at 37° C. for one hour, all added solutions were removed and replaced with 200 μl of RPMI culture solution. Thereafter, the cytopathic effect (CPE) was checked every day by culturing at 37°C, and in each well, the CPE was confirmed after 5 days of culturing. In wells without CPE, virus growth was confirmed by staining with SDOW 17 antibody. Neutralizing antibody titer was measured by calculating the reciprocal maximum dilution factor without evidence of virus growth, and the measurement results are shown in Table 11 below and in FIG. 5.

As shown in Table 11 and FIG. 5, it was possible to confirm that the LMY+BP2017 chimeric virus resulted in a significantly higher secretion of neutralizing antibodies by the cells compared to the original LMY strain.

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Sequence listing

<110> BioPoA, Inc.

REPUBLIC OF KOREA(Animal and Plant Quarantine Agency)

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tatttggcct cccggctgcc catgctacac aacctgcgca tgacaggttc aaatgtaacc 540

atagtgtata atagcacttt gaatcaggtg tttgctattt ttccaacccc tggttcccgg 600

ccaaagcttc atgattttca gcaatggtta atagctgtac attcctccat attttcctct 660

gttgcagctt cttgtactct ttttgttgtg ctgtggttgc gggttccaat actacgtact 720

gtttttggtt tccgctggtt aggggcaatt tttctttcga actcacagtg aattacacgg 780

tgtgtccacc ttgcctcacc cggcaagcag ccacagagat ctacgaaccc ggtaggtctc 840

tttggtgcag gatagggtat gaccgatgtg aggaggatga tcatgacgag ctagggttta 900

tggtaccgcc tggcctctcc agcgaaggcc acttgactag tgtttacgcc tggttggcgt 960

tcttgtcctt cagctacacg gcccagttcc atcccgagat attcgggata gggaatgtga 1020

gtcgagttta tgttgacatc aaacatcaac tcatctgcgc cgaacatgac gggcagaaca 1080

ccaccttgcc tcgtcatgac aacatttcag ccgtgtttca gacctattac caacatcaag 1140

tcgacggcgg caattggttt cacctagaat ggcttcgtcc cttcttttcc tcgtggttgg 1200

ttttaaatgt ctcttggttt ctcaggcgtt cgcctgcaaa ccatgtttca gttcgagtct 1260

tgcagatatt aagaccaaca ccaccgcagc ggcaagcttt gctgtcctcc aagacatcag 1320

ttgccttagg catcgcgact cggcctctga ggcgattcgc aaaatccctc agtgccgtac 1380

ggcgataggg acacccgtgt atgttaccat cacagccaat gtgacagatg agaattattt 1440

acattcttct gatctcctca tgctttcttc ttgccttttc tatgcttctg agatgagtga 1500

aaagggattt aaggtggtat ttggcaatgt gtcaggcatc gtggctgtgt gtgtcaattt 1560

taccagctac gtccaacatg tcaaggagtt tacccaacgc tccctggtgg tcgaccatgt 1620

gcggttgctc catttcatga cacctgagac catgaggtgg gcaactgttt tagcctgtct 1680

ttttgccatt ctgttggcaa tttgaatgtt taagtatgtt ggagaaatgc ttgaccgcgg 1740

gctgttgctc gcaattgctt tctttgtggt gtatcgtgcc gttctgtttt gctgtgctcg 1800

ccaacgccag caacgacagc agctcccatc tacagctgat ttacaacttg acgctatgtg 1860

agctgaatgg cacagattgg ctagctaaca aatttgattg ggcagtggag agttttgtca 1920

tctttcccgt tttgactcac attgtctcct atggtgccct cactaccagc catttccttg 1980

acacagtcgc tttagtcact gtgtctaccg ccgggtttgt tcacgggcgg tatgtcctaa 2040

gtagcatcta cgcggtctgt gccctggctg cgttgacttg cttcgtcatt aggtttgcaa 2100

agaattgcat gtcctggcgc tacgcgtgta ccagatatac caactttctt ctggacacta 2160

agggcggact ctatcgttgg cggtcgcctg tcatcataga gaaaaggggc aaagttgagg 2220

tcgaaggtca tctgatcgac ctcaaaagag ttgtgcttga tggttccgtg gcaaccccta 2280

taaccagagt ttcagcggaa caatggggtc gtccttagat gacttctgtc atgatagcac 2340

ggctccagaa aaggtgcttt tggcgttttc tattacctac acgccagtga tgatatatgc 2400

cctaaaggtg agtcgcggcc gactgctagg gcttctgcac cttttgatct tcctgaattg 2460

tgctttcacc ttcgggtaca tgactttcgc gcactttcag agtacaaata aggtcgcgct 2520

cactatggga gcagtagttg cactcctttg gggggtgtac tcagccatag aaacctggaa 2580

attcatcacc tccagatgcc gtttgtgctt gctaggccgc aagtacattc tggcccctgc 2640

ccaccacgtt gaaagtgccg caggctttca tccgattgcg gcaaatgata accacgcatt 2700

tgtcgtccgg cgtcccggct ccactacggt caacggcaca ttggtgcccg ggttaaaaag 2760

cctcgtgttg ggtggcagaa aagctgttaa acagggagtg gtaaaccttg tcaaatatgc 2820

caaataacaa cggcaagcag cagaagagaa agaaggggga tggccagcca gtcaatcagc 2880

tgtgccagat gctgggtaag atcatcgctc agcaaaacca gtccagaggc aagggaccgg 2940

gaaagaaaaa taagaagaaa aacccggaga agccccattt tcctctagcg actgaagatg 3000

atgtcagaca tcactttacc cctagtgagc ggcaattgtg tctgtcgtca atccagaccg 3060

cctttaatca aggcgctgggg acttgcaccc tgtcagattc agggaggata agttacactg 3120

tggagtttag tttgcctacg catcatactg tgcgcctgat ccgcgtcaca gcatcaccct 3180

cagcatga 3188

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> [A]n

<400> 3

aaaaaaaaaaaaaaaaaaaaaaa 24

<210> 4

<211> 15443

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric LMY-BP2017 virus sequence

<400> 4

atgacgtata ggtgttggct ctatgccttg acatttgtat tgtcaggagc tgtgaccatt 60

ggcacagtcc aaaacttgct gcgcagaaac acccttctgc gacagcctcc ttcaggggag 120

cttggggtct atccctagca ccttgcttct ggagttgcac tgctttacgg tctctccacc 180

cctttaacca tgtctgggat acttgatcgg tgcacgtgta cccccaatgc cagggtgttt 240

atggcgggagg gccaagttta ctgcacacga tgtctcagtg cacggtctct ccttcccctg 300

aatctccaag tttctgagct cggggtgctg ggcctattct acaggcccga agagccactc 360

cggtggacgt tgccacgtgc attccccact gttgagtgct cccccgccgg ggcctgctgg 420

ctttctgcaa tctttccaat tgcacgaatg accagtggaa acctaaactt ccaacaaaga 480

atggtacggg tcgcagctga gatctacaga gttggccagc tcacccctgc agttttgaag 540

gctctacaag tttatgagcg gggttgccgc tggtatccca ttgttggacc tgtccctgga 600

gtggccgttt tcgccaactc cctgcatgtg agtgatagac ctttcccggg agcaactcac 660

gtattaacca acctgccact cccgcagaga cccaagcctg aggacttttg ccccttcgag 720

tgtgctatgg ctgctgtcta cgatgttggt catggcgccg tcatgtatgt ggccgatgag 780

agagtctcct gggcccctcg cggcggggac gaagttagat tcgaaacggt cccacaggag 840

cttaagtcgg ttgcgaacca actctgcacg tcgttcccac cccaccacgt agtcgatatg 900

tctaagttcg cctttaccgc ccccggttgc ggcgtatcta tgcgggtcga acgtcaatac 960

ggctgtctcc ccgccgatac ggtccccgaa ggcaactgct ggtggagctt gttcgattcg 1020

ctcccactcg aagtgcaagg caaagagatt cgccacgcta accaattcgg gtatcagact 1080

aagcatggcg tatccggtaa gtacctacag cggaggctgc aaattaacgg tctccgcgca 1140

gtcgctgacc ctaacggacc tttcgtcgta cagtacttct ccgtcaagga gagttggatc 1200

1260

agggttgagt ctaacacgtc accattggct aacaaggacg aaaaaatttt ccggtttggc 1320

agtcataagt ggtacggcgc tggtaagaga gcaaggaaag cacgctctcg tgcgactaat 1380

acagtcgctg accgcgcttt gtccgttcgt gaaatctggc aggccaagga gcatgaggtt 1440

accggcgcca ataaggctga gcacctcaaa cactactccc cacctgccga agggaattgt 1500

ggttggcact gcatttccgc catcgtcaac cggatggtta attccaaatt tgaaaccacc 1560

cttcccgaaa gagtgagacc tccagatgac tgggctaccg acgaggatct tgcgaatgcc 1620

atccaaatcc tcagactccc tgcggcctta gacaggaacg gtgcttgtgc tagcgccaag 1680

tacgtactta agctggaagg tgagcattgg actgtcactg tgacccctgg gatgtcccct 1740

tctttgctcc ctcttgaatg tgttcaggggc tgttgtgggc acaagggcgg tcttggttcc 1800

cggacgcag tcgaggtctc cggatttgac cctgcctgcc ttgaccgtct ggctgaggtg 1860

atgcacctgc ctagcagtgc tatcccagcc gctctggccg aaatgtctgg tgattccgat 1920

cgttcggctt ctccggtcac caccgtgtgg actgtttcgc agttctttgc ccgtcacagc 1980

ggaggaaatc accctgacca agtgcgctta gggaaaatca tcagcctttg tcaggtgatt 2040

gaggactgct gctgttccca gaacaaaacc aaccgggtca ccccggagga ggttgcagca 2100

aagattgacc tgtacctccg cggtgcaaca aatcttgaag aatgcttggc caggctcgag 2160

aaagcgcgcc cgccacgggt aatcgacacc ttctttgatt gggacgttgt gctccctggg 2220

gttgaggcag caacccaaac gaccaagctg ccccaggtca accagtgtcg tgctctggtc 2280

ccctgttgtga ctcaaaagtc cttggacaac aactcggtcc ccctgaccgc cttttcactg 2340

gctaactact actaccgcgc gcaaggtgac gaagtttgtc accgtgaaag acttaatgcc 2400

gtgctttcca agctggaaga ggttgtccga gaagagtacg ggctcatgcc taccggggcct 2460

ggccccggc ccacattgcc acgcgggctc gacgaactca aagaccagat ggaggtggac 2520

ttgctgaaac tggctaacgc ccaggcgact tctgacatga tggcttgggc agtcgagcag 2580

gttgacctaa aaacttgggt caagaactac ccgcggtgga tgccaccacc tcctccgcca 2640

aaagttcggc ctcgaaaaac gaagcctgtc aagagcttgc cagagggaaa gcccgccccc 2700

gcccctcgca ggagggttgg gactggttgt ggcagcccga cttcattggg cgatgatgtt 2760

cttaacagtt gggaagattt ggctgttggc agcccctcgg gtctcccgac cccccctgag 2820

ccggcaacac cttcgagtga gccagtggtt gtgtcagcac cgcagtgcat cttcagaccg 2880

gcgacgccct tgagtgagcc gaccccaatt cccgcacccc gtgggactgc gtctcgacca 2940

gtgacacctt tgagcgagcc gatccctgtg cctgcaccgc ggcgtaaatt tcagcaggcg 3000

agaaagttga gttcggcggt ggtaattccg ccgtaccagg acgagcccct ggatttgtcc 3060

gcttcctcgc tgactgaata tgaagccctt cctctagcgc cgccgcaaag cgagggcgct 3120

ctaagagtgg aggggcatga agttgaggaa gccctgagtg aaatctcaga catgtcgggt 3180

ggcactaaac ctgcgcccgt atcatcaagc agctccttgt ccagcgtgag aatcacacgc 3240

ccaaaacact cagctcaagc catcatcgac tcggtcgggc cctgcagtgg gcatctccaa 3300

gaggtgaagg agatatgcct tagtgtcatg cgcgaggcat gtgatgcgac taagctcaat 3360

gaccccgcta cgcaggaatg gctctctcgc atgtgggatc gggtggacat gctgacttgg 3420

cgcaacacgt ctgcttacca gacgcttagc accttaggtg gctggtcaag gttcctccca 3480

3540

cctgcacctt ccgtgggtgc ggagagcgac ctcaccattg gctcagttgc tactgaggat 3600

3660

gccttctccg aggataaacc ggtggatgac caaccttccg aagacctccg gacatcgtcg 3720

caaggacctg acgggagcac atcagctccg tccgcagata cagctggcgc cggctcatcc 3780

accgatttgc cgtcttcagg cggcgtggat gcggacggag ggggaccgct tcgggcggta 3840

aaaagaaaaa ccgaacggct ctttgaccaa ctgagtcgtc aggtttttga cctcgtctcc 3900

catctcccta ttttcttctc acgcctcttc aaccctggcg gtggttactc tccgggtgat 3960

tggggttttg cagcttttac tctattgtgc ctctttttat gttacagtta cccagcattt 4020

ggcatcgttc ccctcttggg tgtattttct gggtcttctc ggcgcgtgcg aatgggggtt 4080

tttggctgct ggttggcttt tgctgttggt ctgttcaagc ctgtgtccga cccagtcggc 4140

gctgcttgtg agtttgactc gccagaatgc agaaacatcc ttcattcttt tgagcttctc 4200

aaaccttggg accctgttcg cagccttgtt gtgggccccg tcggtctcgg ttttgccatt 4260

cttggcaggc tactggggcgg ggcacgcagc atctggcact ttttgcttag gcttggcatt 4320

gttgcagact gtgtcttggc tggagcttat gtgctttctc aaggtaggtg caaaaagtgt 4380

tggggatctt gtataagaac cgctcctaat gaggtcgctt tcaacgtgtt tccttttacg 4440

cgtgcaacca ggtcgtcact tgttgacctt tgcgatcggt tttgtgcgcc gagaggcatg 4500

gaccccattt ttctcgccac tgggtggcgt ggatgctggg ccggtcggag ccccattgag 4560

caaccctctg aaaaacccat cgcgtttgcc caattggatg aaaagaagat tacggctagg 4620

actgtggtcg cccagcctta tgatcccaac caagccgtaa agtgcctgcg ggtattgcag 4680

gcgggagggg cgatggtggc tgaggcggtc ccaaaagtgg tcagggtttc cgctgttcca 4740

tttcgagccc ccttttttcc taccggagtg aaggttgacc ctgaatgtag agtagtggtt 4800

gaccctgaca ctttcactgc agctctccgg tctggttact ccaccacaaa cctcgtcctc 4860

ggtgtggggg actttgccca gctgaatgga ttaaaaatca ggcaaatttc taagccttca 4920

ggggggggcc cacatctcat ggctgccctg catgttgcct gctcgatgat tttgcacatg 4980

tttgctggga tttatgtgac tgcggtgggt tcttgcggca ccggcaccaa cgatccgtgg 5040

tgcgcaaacc catttgccgt ccctggctac ggacctggct ccctctgcac gtccagattg 5100

tgtatttccc agcacggcct taccctgccc ttgacagcac ttgtggcggg attcggcatc 5160

caggaaattg ccttagtcgc tttgatcttt gtttccatcg gaggcattgc tcataggttg 5220

agttgtaaag ctgacatgct gtgtgttttg cttgcaattg ccagctatgt ttgggtacct 5280

cttacctggt tactttgtgt ttttccttgc tggttgcgct ggttttcttt gcatcccctt 5340

accgtcctat ggttggtgtt tttcttgatt tctgtgaata tgccttcagg aatcttggcc 5400

atggtgttgt tagtttctct ttggcttctt ggtcgttata ctaatgtcgc cggtcttgtc 5460

actccctacg acattcatca ttacaccagt ggcccccgcg gtgttgccgc attggctact 5520

gcaccagatg gaacctactt agccgctgtt cgccgcgctg cgttgactgg ccgcaccatg 5580

ttgttcaccc cgtcccagct tgggtctctt cttgagggcg ctttcagaac tcgaaagccc 5640

tcactgaaca ccgtcaatgt ggtcgggtcc tccatgggct ctggcggggt gtttaccatt 5700

gacgggaaag tcaggtgcgt gactgccaca catgtcctta cgggtaactc agccagggtt 5760

tccggaaccg gcttcaatca aatgcttgac tttgacgtaa aaggggattt cgctatagcc 5820

gattgcccga attggcaagg ggctgccccc aaaacccaat tctgcgagga tggatggact 5880

ggccgtgcct attggctaac atcctctggc gtcgaacccg gcgtcattgg gaaaggattc 5940

gccttctgct tcaccgcgtg cggcgattcc gggtccccag tgatcaccga ggccggtgag 6000

cttgtcggtg ttcacacgggg atcgaataaa caaggggggg gtattgtcac acgcccctca 6060

ggccagtttt gtaatgtggc acccatcaag ctgagcgaat taagtgaatt ctttgctggg 6120

6180

gtgccttcag atctttgtgc cttgcttgct gccaaacctg aactggaagg aggtctctcc 6240

accgtccaac ttctgtgtgt gttttttcctt ctgtggagaa tgatgggaca tgcctggacg 6300

6360

agtattttct cctttggaat gtttgtgcta tcctggctca cgccatggtc tgcgcaagtt 6420

ctgatgatca ggctcctgac agcagctctt aacaggaata gatgttcact tgcctttttc 6480

agcctcggtg cagtgaccgg ttttgttgca gatcttgcgg ctactcaggg gcatccgttg 6540

caggcagtga tgaatttgag cacctatgca ttcctgcctc gggtaatggt tgtgacttca 6600

ccagtcccag tgatcgcgtg tggtgttgtg cacctactcg ccatcattct gtacctgttt 6660

aaataccgta gcctgcacca tatccttgtt ggcgatggag tgttctcttc ggccttcttc 6720

ctgcgatatt ttgccgaagg aaagttgagg gaaggggtgt cacaatcctg cggaatgaat 6780

catgagtctc tgactggtgc cctcgctatg agactcaatg acgaggactt ggatttcctc 6840

atgaaatgga ctgattttaa gtgcttcgtt tctgcgtcca acatgaggaa tgcagcgggt 6900

caatttatcg aggctgcata tgctaaagca cttagagttg aactggccca gttggtgcag 6960

7020

caactctcgc ccggtgatat tgttgtcgct ctcggccaca cgcctgttgg cagtatcttc 7080

gacttaaagg ttggtagcac caagcatacc ctccaagcca ttgagaccag ggttcttgct 7140

gggtccaaaa tgaccgtggc gcgcgtcgtc gacccgaccc ccacgccccc acccgcgccc 7200

gtgcccatcc ccctcccacc gaaagttctg gagaatggcc ccaacgcttg gggggatgag 7260

gaccgtttga ataagaagaa aagacgcagg atggaagccc tcggcatcta tgttatgggc 7320

gggaaaaagt accagaaatt ttgggacaag aactccggtg atgtgtttta tgaggaggtc 7380

cataacaaca cagatgagtg ggagtgtctc acagttggcg accctgccga ctttgaccct 7440

gagaagggaa ccctgtgtgg acatgtcacc attgacaaca aggcttacca tgtttacacc 7500

tcctcatctg gtaagaagtt cttggttccc gtcaacccag agaacggaag agttcaatgg 7560

gaagctgcaa agctttccgt ggagcaggcc ctaggcatga tgaatgtcga cggcgaactg 7620

actgccaagg aactggagaa actgaaaaga ataattgaca aactccaggg cctgactaag 7680

gagcagtgtt taaactgcta gccgccagcg acttgacccg ctgtggtcgc ggcggcttgg 7740

ttgttactga gacagcggtg aaaatagtca aatttcacaa tcggactttc actctggggc 7800

ctgtgaattt aaaagtggcc agtgaggttg agctaaaaga tgcggttgag cacaaccaac 7860

acccggttgc gaggccgatc gatggtggag ttgtgctcct gcgttccgcg gttccctcgc 7920

ttatagacgt cttgatctcc ggtgctgaca catctcccaa gttacttgcc cttcacgggc 7980

cgggaaacac tgggatcgat ggcacgctct gggatttcga gtccgaagcc actaaagagg 8040

aagtcgcact cagtgcgcaa ataatacagg cttgtgacat taggcgtggc gacgctcctg 8100

aaattggtct cccttataag ctgtaccctg ttaggggcaa ccctgagcgg gtgaaaggag 8160

ttctgcagaa tacaaggttt ggagacatac cctacaaaac ccctagtgac accggggagcc 8220

cagtgcacgc ggctgcctgc cttacgccca acgccactcc tgtgactgat gggcgttccg 8280

8340

tccttgatta ccttgactct aggcctgact gccctaaaca gctgacagag cacggctgcg 8400

aagacgccgc actgaaagac ctctctaaat atgacttatc tacccaaggc tttgtcttac 8460

ctggggttct tcgccttgtg cggaaatacc tgtttgccca tgtaggtaaa tgtccacccg 8520

ttcatcggcc ttctatttac ccgctaaga attctatggc tggaataaat gggaacaggt 8580

tcccaaccaa agacatccag agcgttcctg aaatcgacgt tctgtgcgca caggctgtgc 8640

gggaaaactg gcaaactgtc accccttgta ctcttaagaa acagtattgc gggaagaaga 8700

agaccaggac catactcggc accaataact tcattgcact ggcccaccga gcagtgttga 8760

gtggtgtcac ccagggcttc atgaaaaagg cgttcaactc gcccatcgcc ctcggaaaga 8820

acaagttcaa ggagctacag actccggtcc tgggcaggtg ccttgaagct gacctcgcat 8880

cctgtgatcg atccacgcct gcaattgttc gctggtttgc tgccaacctt ctttatgaac 8940

ttgcctgtgc tgaagagtat ctaccgtcgt atgtgctgaa ctgctgccac gacttactag 9000

tcacgcagtc cggcgcagtg actaagagag gtggcctgtc gtctggcgac ccgatcacct 9060

ctgtgtctaa caccatttac agtttggtga tctatgcaca gcacatggtg cttagttact 9120

ttaaaagtgg tcacccccat ggccttctgt tcttacaaga ccagctaaag tttgaggaca 9180

tgctcaaggt tcaacccctg atcgtctatt cggacgatct cgtgctgtat gccgagtctc 9240

ccaccatgcc aaattatcac tggtgggttg agcatctgaa tttgatgctg gggtttcaga 9300

cggacccaaa aaaaacagca ataacagatt cgccatcatt tctgggctgt agaataatga 9360

atgggcgcca gctagtcccc aaccgtgaca ggatcctcgc ggccctcgcc tatcacatga 9420

aggcaagtaa tgtttctgaa tactatgcct cagcggctgc aatactcatg gacagctgtg 9480

cttgtttgga gtatgatcct gaatggtttg aagaacttgt agttggaata gcgcagtgcg 9540

cccgcaagga tggctacagc tttcccggca cgccgttctt catgtccatg tgggaaaaac 9600

tcaggtccaa ttatgagggg aagaagtcga gagtgtgcgg gtactgcggg gccccggccc 9660

cgtacgctac tgcctgtggc ctcgacgtct gcatctacca cacccacttc caccagcatt 9720

gtccagtcac aatctggtgt ggccatccag cgggttctgg ttcttgtagt gagtgcaaat 9780

cccctgtagg gaaaggcaca aaccctttag acgaggtgct ggaacaagtc ccgtataagc 9840

ccccacggac cgttatcatg catgtagagc agggtctcac cccccttgat ccaggtagat 9900

accaaactcg ccgcggatta gtttccgtta ggcgtggaat taggggaaat gaagttgaac 9960

taccagacgg tgattatgct agcaccgcct tgctccctac ctgcaaagag atcaacatgg 10020

tcgctgtcgc ttccaatgta ttgcgcagca ggttcatcat cggcccaccc ggtgcaggga 10080

aaacatactg gctccttcaa caggtccagg acggtgatgt tatttacaca ccaactcacc 10140

agaccatgct tgacatgatc agggctttgg ggacgtgccg gttcaacgtc ccggcaggca 10200

caacgctgca attccctgtc ccctcccgca ccggtccgtg ggttcgcatc ctggccggcg 10260

gttggtgtcc tggcaagaat tccttcctag atgaagcagc gtattgcaac caccttgatg 10320

ttttgaggct tcttagcaaa actaccctca cctgtctagg agacttcaag caactccacc 10380

cagtgggttt tgattctcat tgttatgttt ttgacatcat gcctcaaact caactgaaga 10440

ccatctggag gttcggacag aatatctgtg atgccatcca gccagattac agggacaagc 10500

10560

aggtcctcac cccctaccac agggaccgag aggacgacgc catcactatt gactccagtc 10620

aaggcgccac attcgatgtg gtcacattgc atttgcccac taaagattca ctcaacaggc 10680

aaagagccct tgttgccatc accagggcta gacacgctat ctttgtgtat gacccacaca 10740

ggcagctgca gagcttgttt gatcttcctg caaaaggcac acccgtcaac cttgcagtgc 10800

accgcgacgg gcagctgatc gtacttgata gaaataacaa agaatgcacg gttgctcagg 10860

ctctaggcaa cggggataaa ttcaggggcca cagacaagcg tgttgcagat tcactccgcg 10920

ccatttgtgc tgatctagaa gggtcgagtt ctccgctccc caaggtcgca cacaacttgg 10980

ggttttattt ctcacctgac ttaacacaat ttgccaaact cccagtggaa cttgcacctc 11040

attggcccgt ggtgacaacc cagaacaatg aaaagtggcc agatcggctg gtcgccagcc 11100

ttcgccctat ccataaatac agccgcgcgt gcatcggtgc cggctatatg gtgggccctt 11160

cggtgtttct aggcactcct ggggtcgtat catactttct tacaaaattt gttaaaggcg 11220

aggctcaaat gcttccagaa acagtcttca gcaccggccg aattgaggtg gattgccggg 11280

aatatcttga cgatcgggag cgggaagttg ctgcatccct cccacacgcc tttattggcg 11340

acgtcaaagg caccaccgtt ggcggatgtc atcatgtcac ctccaggtac cttcctcgct 11400

tccttcccaa agagtcagtt gcggtagtcg gggtctcaag ccccgggaaa gccgcgaagg 11460

caatgtgcac actgacagat gtgtacctcc cagatcttga agcctatctc cacccggaga 11520

cccagtccag gtgctggaaa atgatgttgg acttcaaaga agttcgactg atggtctgga 11580

11640

gttatgcctc gtacgtccgt gttcctgtca actccacggt gtacttggac ccctgcatgg 11700

gcccagccca ttgcaacaga cgagttgtcg ggtccactca gtggggagct gacctcgcaa 11760

tcacccccta tgattatggc gccaagatca ttttgtctag cgcatatcac ggtgaaatgc 11820

cccctgggta caagattctg gcgtgtgcgg agttctcatt ggatgacccg gtcagataca 11880

11940

aggattggga agattataat gatgcgtttc gtgcgcgcca gaaagggaaa atttataagg 12000

ccactgccac cagcttgaag ttttatttcc ctccgggccc tgtcgttgaa ccaactttgg 12060

gcctgaactg aaggcgcgcc atgaaatggg gtccatgcaa agcctttttt acaaaattgg 12120

ccaacttttt gtggatgctt tcacggagtt cttggtgtcc attgttgata tcattatatt 12180

tttggccatt ttgtttggct tcaccatcgc cggttggctg gtggtctttt gcatcagatt 12240

ggtttgctcc gcgatactcc gtacgcgccc tgccattcac tctgagcaat tacagaagat 12300

cttatgaggc ctttctttcc cagtgccaag tggacattcc cacctgggga actaaacatc 12360

ctttggggat gctttggcac cataaggtgt caaccctgat tgatgaaatg gtgtcgcgtc 12420

gaatgtaccg catcatggaa aaagcagggc aggctgcctg gaaacaggtg gtgagcgagg 12480

ctacgctgtc tcgcattagt agtttggatg tggtggctca ttttcagcat ctagccgcca 12540

ttgaagccga gacctgtaaa tatttggcct cccggctgcc catgctacac aacctgcgca 12600

tgacaggttc aaatgtaacc atagtgtata atagcacttt gaatcaggtg tttgctattt 12660

ttccaacccc tggttcccgg ccaaagcttc atgattttca gcaatggtta atagctgtac 12720

12780

gggttccaat actacgtact gtttttggtt tccgctggtt aggggcaatt tttctttcga 12840

actcacagtg aattacacgg tgtgtccacc ttgcctcacc cggcaagcag ccacagagat 12900

12960

tcatgacgag ctagggttta tggtaccgcc tggcctctcc agcgaaggcc acttgactag 13020

13080

attcgggata gggaatgtga gtcgagttta tgttgacatc aaacatcaac tcatctgcgc 13140

cgaacatgac gggcagaaca ccaccttgcc tcgtcatgac aacatttcag ccgtgtttca 13200

gacctattac caacatcaag tcgacggcgg caattggttt cacctagaat ggcttcgtcc 13260

13320

ccatgtttca gttcgagtct tgcagatatt aagaccaaca ccaccgcagc ggcaagcttt 13380

gctgtcctcc aagacatcag ttgccttagg catcgcgact cggcctctga ggcgattcgc 13440

aaaatccctc agtgccgtac ggcgataggg acacccgtgt atgttaccat cacagccaat 13500

gtgacagatg agaattattt acattcttct gatctcctca tgctttcttc ttgccttttc 13560

13620

13680

tccctggtgg tcgaccatgt gcggttgctc catttcatga cacctgagac catgaggtgg 13740

gcaactgttt tagcctgtct ttttgccatt ctgttggcaa tttgaatgtt taagtatgtt 13800

ggagaaatgc ttgaccgcgg gctgttgctc gcaattgctt tctttgtggt gtatcgtgcc 13860

gttctgtttt gctgtgctcg ccaacgccag caacgacagc agctcccatc tacagctgat 13920

ttacaacttg acgctatgtg agctgaatgg cacagattgg ctagctaaca aatttgattg 13980

ggcagtggag agttttgtca tctttcccgt tttgactcac attgtctcct atggtgccct 14040

14100

14160

cttcgtcatt aggtttgcaa agaattgcat gtcctggcgc tacgcgtgta ccagatatac 14220

caactttctt ctggacacta agggcggact ctatcgttgg cggtcgcctg tcatcataga 14280

gaaaaggggc aaagttgagg tcgaaggtca tctgatcgac ctcaaaagag ttgtgcttga 14340

tggttccgtg gcaaccccta taaccagagt ttcagcggaa caatggggtc gtccttagat 14400

gacttctgtc atgatagcac ggctccagaa aaggtgcttt tggcgttttc tattacctac 14460

acgccagtga tgatatatgc cctaaaggtg agtcgcggcc gactgctagg gcttctgcac 14520

cttttgatct tcctgaattg tgctttcacc ttcgggtaca tgactttcgc gcactttcag 14580

agtacaaata aggtcgcgct cactatggga gcagtagttg cactcctttg gggggtgtac 14640

tcagccatag aaacctggaa attcatcacc tccagatgcc gtttgtgctt gctaggccgc 14700

aagtacattc tggcccctgc ccaccacgtt gaaagtgccg caggctttca tccgattgcg 14760

gcaaatgata accacgcatt tgtcgtccgg cgtcccggct ccactacggt caacggcaca 14820

ttggtgcccg ggttaaaaag cctcgtgttg ggtggcagaa aagctgttaa acagggagtg 14880

gtaaaccttg tcaaatatgc caaataacaa cggcaagcag cagaagagaa agaaggggga 14940

tggccagcca gtcaatcagc tgtgccagat gctgggtaag atcatcgctc agcaaaacca 15000

15060

tcctctagcg actgaagatg atgtcagaca tcactttacc cctagtgagc ggcaattgtg 15120

tctgtcgtca atccagaccg cctttaatca aggcgctggg acttgcaccc tgtcagattc 15180

agggaggata agttacactg tggagtttag tttgcctacg catcatactg tgcgcctgat 15240

ccgcgtcaca gcatcaccct cagcatgatg ggctggcatt cttgaggcat ctcagtgttt 15300

gaattggaag aatgtgtggt gaatggcact gattgacatt gtgcctctaa gtcacctatt 15360

caattagggc gaccgtgtgg gggtgagatt taattggcga gaaccatgcg gccgaaatta 15420

aaaaaaaaaaaaaaaaaaa 15443

<210> 5

<211> 609

<212> DNA

<213> Artificial Sequence

<220>

<223> NSP1-beta sequence LMY

<400> 5

gctgctgtct acgatgttgg tcatggcgcc gtcatgtatg tggccgatga gagagtctcc 60

tgggcccctc gtggcgggga tgaagtaaga tttgaaactg tcccacagga gctcaagtcg 120

gttgcgaacc aactctgcac ctccttccca ccccaccacg tagtggacat gtctaagttc 180

gcctttacag cccctgggtg tggtgtttct atgcgggtcg aacgtcaata tggctgtctc 240

cccgctgaca ctgtccccga aggcaactgc tggtggagct tgtttgactc gctcccattg 300

gaagtccagg gcaaagaaat tcgccatgct aaccaatttg gctaccagac caagcatggt 360

gtctctggta agtacctaca gcggaggctg caaattaatg gtctccgagc agtagctgac 420

ccaaatggac ctttcgtcgt acagtacttc tccgtcaagg agagttggat ccgccacttg 480

aaactagcgg aagaacccag ttaccctggg tttgaggacc tcctcagaat aagggttgag 540

tctaacacgt caccattggc taacaaggat gaaaaaattt tccggtttgg cagtcataag 600

tggtacggc 609

<210> 6

<211> 609

<212> DNA

<213> Artificial Sequence

<220>

<223> NSP1-beta sequence LMY ver2

<400> 6

gccgccgtat acgacgtcgg acatggcgcc gttatgtacg ttgccgacga gagagtctcc 60

tgggcccctc gcggcggcga cgaagttaga ttcgaaacgg tcccacagga gcttaagtcg 120

gttgcgaacc aattatgtac gtcgttccca ccccaccacg tagtcgatat gtctaagttc 180

gcctttaccg cccccggttg cggcgtatct atgcgggtcg aacgtcaata cggctgtctc 240

cccgccgata cggtccccga aggcaactgt tggtggagct tgttcgattc gctcccactc 300

gaagtgcaag gcaaagagat tcgccacgct aaccaattcg ggtatcagac taagcatggc 360

gtatccggta agtacctaca gcgtaggctg caaatcaacg gtctccgcgc agtcgctgac 420

cctaacggac ctttcgtcgt acagtacttc tccgtcaagg agagttggat ccgccacttg 480

aaactggccg aagaacctag ttacccgggg ttcgaggacc tcctccgcat aagggttgag 540

tctaatacgt caccattggc taacaaggac gaaaaaattt tccggtttgg cagtcataag 600

tggtacggc 609

<210> 7

<211> 8

<212> DNA

<213> Artificial Sequence

<220>

<223> AscI restriction enzyme recognition site

<400> 7

ggcgcgcc 8

<210> 8

<211> 8

<212> DNA

<213> Artificial Sequence

<220>

<223> PacI restriction enzyme recognition site

<400> 8

ttaattaa 8

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Direct PCR primer for LMY DNA amplification

<400> 9

atgacgtata ggtgttggct cta 23

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Reverse PCR primer for LMY DNA amplification

<400> 10

ctgaggattt ggatggcatt 20

<210> 11

<211> 1149

<212> DNA

<213> Artificial Sequence

<220>

<223> NSP1 LMY gene

<400> 11

atgtctggga tacttgatcg gtgcacgtgt acccccaatg ccagggtgtt tatggcggag 60

ggccaagttt actgcacacg atgtctcagt gcacggtctc tccttcccct gaatctccaa 120

gtttctgagc tcggggtgct gggcctattc tacaggcccg aagagccact ccggtggacg 180

ttgccacgtg cattccccac tgttgagtgc tcccccgccg gggcctgctg gctttctgca 240

atctttccaa ttgcacgaat gaccagtgga aacctaaact tccaacaaag aatggtacgg 300

gtcgcagctg agatctacag agttggccag ctcacccctg cagttttgaa ggctctacaa 360

gtttatgagc ggggttgccg ctggtatccc attgttggac ctgtccctgg agtggccgtt 420

ttcgccaact ccctgcatgt gagtgataga cctttcccgg gagcaactca cgtattaacc 480

aacctgccac tcccgcagag acccaagcct gaggactttt gccccttcga gtgtgctatg 540

gctgctgtct acgatgttgg tcatggcgcc gtcatgtatg tggccgatga gagagtctcc 600

tgggcccctc gtggcgggga tgaagtaaga tttgaaactg tcccacagga gctcaagtcg 660

gttgcgaacc aactctgcac ctccttccca ccccaccacg tagtggacat gtctaagttc 720

gcctttacag cccctgggtg tggtgtttct atgcgggtcg aacgtcaata tggctgtctc 780

cccgctgaca ctgtccccga aggcaactgc tggtggagct tgtttgactc gctcccattg 840

gaagtccagg gcaaagaaat tcgccatgct aaccaatttg gctaccagac caagcatggt 900

gtctctggta agtacctaca gcggaggctg caaattaatg gtctccgagc agtagctgac 960

ccaaatggac ctttcgtcgt acagtacttc tccgtcaagg agagttggat ccgccacttg 1020

aaactagcgg aagaacccag ttaccctggg tttgaggacc tcctcagaat aagggttgag 1080

tctaacacgt caccattggc taacaaggat gaaaaaattt tccggtttgg cagtcataag 1140

tggtacggc 1149

<210> 12

<211> 1149

<212> DNA

<213> Artificial Sequence

<220>

<223> NSP1 gene LMY ver2

<400> 12

atgtctggga tacttgatcg gtgcacgtgt acccccaatg ccagggtgtt tatggcggag 60

ggccaagttt actgcacacg atgtctcagt gcacggtctc tccttcccct gaatctccaa 120

gtttctgagc tcggggtgct gggcctattc tacaggcccg aagagccact ccggtggacg 180

ttgccacgtg cattccccac tgttgagtgc tcccccgccg gcgcatgctg gctttccgct 240

atctttccaa tcgcacgaat gaccagtgga aacctaaact tccaacaaag aatggtacgg 300

360

gtatacgagc ggggttgtag gtggtatccc atcgtaggac ctgtccctgg agtggccgtt 420

ttcgccaact ccctgcatgt gagtgataga cctttcccgg gagcaactca cgtattaacc 480

aacctgccac tcccgcagag acccaagcct gaggactttt gccccttcga gtgtgctatg 540

gccgccgtat acgacgtcgg acatggcgcc gttatgtacg ttgccgacga gagagtctcc 600

tgggcccctc gcggcggcga cgaagttaga ttcgaaacgg tcccacagga gcttaagtcg 660

gttgcgaacc aattatgtac gtcgttccca ccccaccacg tagtcgatat gtctaagttc 720

gcctttaccg cccccggttg cggcgtatct atgcgggtcg aacgtcaata cggctgtctc 780

cccgccgata cggtccccga aggcaactgt tggtggagct tgttcgattc gctcccactc 840

gaagtgcaag gcaaagagat tcgccacgct aaccaattcg ggtatcagac taagcatggc 900

gtatccggta agtacctaca gcgtaggctg caaatcaacg gtctccgcgc agtcgctgac 960

1020

aaactggccg aagaacctag ttacccgggg ttcgaggacc tcctccgcat aagggttgag 1080

tctaatacgt caccattggc taacaaggac gaaaaaattt tccggtttgg cagtcataag 1140

tggtacggc 1149

<---

Claims (18)

1. Chimeric porcine reproductive and respiratory syndrome virus (PRRSV) that provides immunity against PRRSV infection, containing a polynucleotide consisting of the structure of structural formula 1, where the polynucleotide is the chimeric virus genome: [structural formula 1] 5'-[X]-[Y]-3', where [X] is a gene fragment obtained by processing the genome of the mutant strain LMY ver2, having the registration number 13394 BP, with restriction enzymes AscI and PacI, [Y] is a gene fragment obtained by processing the genome of the mutant strain BP2017-2, having the registration number BP 13393, with AscI and PacI restrictases. 2. Chimeric PRRSV virus according to claim 1, where the polynucleotide additionally contains [A] n at the 3'-end [Y] of structural formula 1, where n is the number of nucleotides with base adenine (A) and an integer from 1 to 100. 3. A chimeric PRRSV virus according to claim 1, wherein the polynucleotide is DNA or RNA. 4. Chimeric PRRSV virus according to claim 1, where [X] contains the NSP1 gene of the mutant strain, where the base is replaced in one or more positions selected from the group consisting of position 222, position 225, position 237, position 240, regulations 252, regulations 306, regulations 309, regulations 312, regulations 315, regulations 324, regulations 327, regulations 330, regulations 333, regulations 336, regulations 339, regulations 342, regulations 345, regulations 357, regulations 363, regulations 366, regulations 378 , regulations 379, regulations 381, regulations 393, regulations 396, regulations 543, regulations 546, regulations 549, regulations 555, regulations 558, regulations 561, regulations 573, regulations 579, regulations 582, regulations 588, regulations 612, regulations 618, regulations 621, regulations 627, regulations 633, regulations 639, regulations 654, regulations 673, regulations 675, regulations 678, regulations 681, regulations 684, regulations 705, regulations 708, regulations 729, regulations 735, regulations 738, regulations 741, regulations 744, provisions 747, p Regulations 771, Regulations 786, Regulations 789, Regulations 792, Regulations 810, Regulations 825, Regulations 828, Regulations 838, Regulations 840, Regulations 846, Regulations 849, Regulations 858, Regulations 867, Regulations 879, Regulations 882, Regulations 885, Regulations 891 , regulations 900, regulations 903, regulations 906, regulations 924, regulations 936, regulations 939, regulations 948, regulations 954, regulations 963, regulations 966, regulations 1026, regulations 1029, regulations 1038, regulations 1047, regulations 1053, regulations 1066, regulations 1068, positions 1086, positions and positions 1110 of the nucleic acid sequence of the NSP1 gene, consisting of the nucleotide sequence of SEQ ID NO:11. 5. The chimeric PRRSV virus according to claim 4, wherein the NSP1 gene of the mutant strain is represented by the nucleic acid sequence of SEQ ID NO:12. 6. The chimeric PRRSV virus according to claim 1, wherein [Y] is the nucleic acid sequence of SEQ ID NO:2. 7. The chimeric PRRSV virus according to claim 1, wherein structural formula 1 is represented by the nucleic acid sequence of SEQ ID NO:4. 8. Chimeric PRRSV virus according to claim 1, having registration number KCTC 13675 BP. 9. Vaccine composition against porcine reproductive and respiratory syndrome virus, containing an effective amount of a chimeric PRRSV virus according to claim 1. 10. The vaccine composition according to claim 9, wherein the vaccine is a live vaccine. 11. The vaccine composition according to claim 9, further comprising one or more selected from the group consisting of carriers, diluents, excipients and adjuvants. 12. The vaccine composition according to claim 9, wherein the 50% cytopathic dose rate (TCID ₅₀ ) of the chimeric virus measured after contacting the porcine lung macrophages with the vaccine composition and its parent strain (GenBank accession number DQ473474.1.) is a TCID ₅₀ index of 0.01 to 0.1 compared to the TCID ₅₀ index of the original strain. 13. The vaccine composition according to claim 9, wherein the index of neutralizing antibodies of the chimeric virus, measured after bringing the porcine lung macrophages into contact with the vaccine composition and its parent strain (registration number in GenBank DQ473474.1.), is from 2 to 8 compared to indicator of neutralizing antibodies of the original strain.

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