KR102276341B1 - Method for producing European porcine reproductive and respiratory syndrome virus and use thereof - Google Patents

Abstract

The present invention relates to a mutant of European porcine reproductive and respiratory syndrome virus into which silent mutants are introduced through codon pair deoptimization at specific positions in genome, and uses thereof. The virus mutant provided by the present invention was attenuated compared to a wild-type strain, and can be used as a vaccine for preventing the European-type porcine reproductive and respiratory syndrome due to reduced proliferation in pigs.

Description

Method for producing European porcine reproductive and respiratory syndrome virus and use thereof

The present invention relates to a method for producing an attenuated mutant of porcine reproductive and respiratory syndrome virus (PRRSV), and the PRRSV mutant prepared by the method provided by the present invention has lower pathogenicity and higher stability than the parent strain.

Porcine Reproductive and Respiratory Syndrome (hereinafter referred to as "PRRS") is a viral disease first discovered in the United States in 1987, and was identified in Asia in the early 1990s following Europe. PRRS is an infectious disease that causes the greatest damage to the domestic pig industry along with swine circovirus infection and foot-and-mouth disease, and is spreading worldwide, causing huge economic losses every year.

PRRS causes impotence, miscarriage, premature birth, and stillbirth in pregnant sows, and causes respiratory symptoms such as sneezing and fever in suckling pigs and finisher pigs. In general, severe respiratory symptoms are caused by secondary infection such as bacteria after being infected with a virus, but in the case of chronic infection, a decrease in weight gain and an increase in mortality rate appear without characteristic clinical symptoms.

The pathogen causing PRRS is the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which belongs to the Arterivirus genus, the Arteriviridae family, and the Nidovirales order. PRRS virus has a positive-sense single stranded RNA (+ssRNA) genome, the size of which is about 15.4 kb, and has been reported to have 9 ORFs. About 80% of the genome is ORF1a and ORF1b, which encode Non Structural Proteins (NSPs), and the remaining 20% are glycosylated structural proteins GP2, GP3, GP4, GP5, and Membrane, M) consists of an ORF encoding a protein, nucleocapsid (N) protein. The minor structural proteins, GP2, GP3, and GP4, form a heterotrimer and act when the virus invades the host cell, and the major structural proteins, GP5 and M, form a heterodimer to infect the virus. acts to increase

The PRRS virus is highly mutated due to the nature of the RNA virus, and thus there is a large genetic difference between PRRS viruses. PRRS virus is largely divided into North American type and European type. Type I (Lelystad virus, LV) representing the European type and Type II representing the Northern American strain ATCC VR2332 (The genome sequence of VR2332 is GenBank registered See number AY150564).

It is known that there is a genetic difference of up to 40% between the North American type and the European type, so that there is no cross-protection. Even among mutants belonging to the same type, cases where cross-protection does not occur have been reported. For this reason, standard mutant-based vaccines have been prepared for each type of virus, but PRRS cannot be effectively prevented due to low cross-protection ability. In order to overcome this, various attempts have been made to produce a vaccine having effective safety, immunogenicity, and protective ability.

Due to these limitations, a lot of effort has been invested in developing a preventive method for this virus for about 30 years after the discovery of the porcine reproductive and respiratory syndrome virus (PRRSV), which is the cause of porcine reproductive and respiratory syndrome (PRRS), which causes enormous damage to the livestock industry. However, effective prevention and management methods have not yet been developed.

Various vaccines, such as inactivated vaccines and live attenuated vaccines, have been developed to control porcine reproductive and respiratory syndrome virus (PRRSV), but only attenuated vaccines with secured infectivity have been found to induce a satisfactory level of protective effect. However, as previously salvaged, because the porcine reproductive and respiratory syndrome virus (PRRSV) is genetically very diverse, cross-immunity between various viruses is absent, so it is difficult to protect various porcine reproductive and respiratory syndrome viruses (PRRSV) with one vaccine. It is difficult. In addition, the current attenuated vaccine can only be produced through successive passages of 100 to 200 or more in cell lines of other animal species, so there is a problem that the development period is long and it is difficult to guarantee efficacy and safety.

The present inventors completed the present invention by preparing attenuated European PRRSV using codon pair deoptimization, or molecular attenuation technology.

The present invention relates to an attenuated PRRSV mutant, a vaccine composition for preventing and/or treating PRRS including the mutant, and a method for preparing the same.

The present invention also provides a method for preventing and/or treating PRRS, comprising administering the vaccine composition.

The attenuated PRRSV mutant strain provided by the present invention is safe as a vaccine strain because its proliferation is reduced compared to the existing virus.

The present invention relates to a method for producing a mutant strain of porcine reproductive and respiratory syndrome (PRRS) virus that can be used as a vaccine. The PRRSV virus mutant prepared by the method of the present invention is attenuated than the parent strain, and thus has low pathogenicity and high stability. Therefore, it can be used as a vaccine for effective prevention and treatment of PRRS disease.

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

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

A “vaccine composition” as used herein may be a PRRS chimeric virus or any immunogenic fragment or fraction thereof, preferably an attenuated PRRS chimeric virus, such as the PRRS chimeric virus of the present invention. It elicits an "immunological response" of the host 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 clinical signs associated therewith.

As used herein, "immune response" refers to any cell- and/or antibody-mediated immune response to the chimeric virus or vaccine administered to an animal receiving the PRRSV chimeric virus of the present invention, or a vaccine composition comprising the same. means Usually, an “immune response” includes, but is not limited to, one or more of the following effects: antibodies, B cells, helper T cells directed specifically against an antigen or antigens included in the composition or vaccine. , production or activation of suppressor T cells and/or cytotoxic T cells and/or γδ T cells. Preferably, the host exhibits a therapeutic or prophylactic immunological response such that resistance to new infections is improved and/or the clinical severity of disease is reduced compared to controls not receiving the immunogenic composition or vaccine. Such prophylaxis will be evidenced by a reduction in frequency or severity, including the absence of and, at most, the symptoms associated with the aforementioned host infection.

As used herein, “pigs”, “pig” and “pig” may be used interchangeably.

"Vaccinate" means administering the PRRSV chimeric virus described herein or a vaccine comprising the same prior to exposure to PRRS disease.

"Prevent" or "prevention" means a decrease in the clinical incidence, severity or frequency of PRRS as a result of administration of the PRRSV virus of the present invention or a vaccine composition comprising the same. The reduction in severity or frequency is a result of comparison with an animal or group of animals not administered with the PRRSV chimeric virus of the present invention or a vaccine composition comprising the same. The animal may preferably be a pig.

The nucleotide sequence of the present specification has been described based on DNA nucleotides for convenience, and when the type of polynucleotide is RNA, all or part of thymine (T) in the nucleotide sequence is substituted with uracil (Uracil, U). it means.

In the present specification, a specific base sequence and/or amino acid "consisting of a sequence" includes all of the sequence including the sequence, essentially including the sequence, and/or consisting of the sequence, and may be appropriately substituted if necessary. can

The present invention provides a mutant of an attenuated porcine reproductive and respiratory syndrome (PRRS) virus, and the mutant may include a silent mutation in the ORF7 region of porcine reproductive and respiratory syndrome virus (PRRSV).

The ORF7 region is an ORF located closest to the 3'-end of the porcine reproductive and respiratory syndrome virus genome, and is known to express the structural protein of PRRSV. If the silent mutation occurs outside the ORF7 site, virus production may not occur. In one embodiment, the introduction of a silent mutation in the gene encoding the NSP1 protein within the ORF1a site results in no virus generation.

The ORF7 sequence of the PRRSV mutant provided by the present invention may have a lower codon pair bias (CPB) than the ORF7 region of wild-type PRRSV. The ORF7 region of the wild-type PRRSV may consist of the nucleic acid sequence of SEQ ID NO: 2.

The codon pair bias may be calculated using a program that can be used for the purpose of calculating CPB known in the art, for example, a Synthetic Attenuated Virus Engineering (SAVE) program, but is not limited thereto.

The codon pair bias is changed when some nucleotide sequences of a gene are substituted with other nucleotides, and is particularly closely related to the proliferation of viruses. Specifically, when CPB levels are reduced, the proliferative capacity of the virus is reduced and attenuated.

The CPB level of the ORF7 region of the attenuated PRRSV mutant strain provided by the present invention is 0.05 to 0.5, 0.05 to 0.3, or 0.05 to 0.2 lower than the CPB value of the ORF7 region of wild-type PRRSV. no.

The CpB and/or UpA levels of the ORF7 region of the attenuated PRRSV mutant provided by the present invention are higher than in the parent strain. Changes in the CpG and UpA ratios are inevitably occurring during the deoptimization process. Increasing the levels of CpG and UpA of eukaryotic genes can cause cell stress and decrease the proliferation of the virus, thereby weakening the virus. In one example, the CpB and / or UpA levels of the attenuated PRRSV mutant provided by the present invention are 1 to 5 times, 1 to 3 times, 1.1 to 5 times, 1.1 to 3 times, 1.3 to 5 times compared to the parent strain. Or it may have a numerical value of 1.3 to 3 times, but is not limited thereto.

In one example, the CpG value of the ORF7 region of the attenuated PRRSV mutant of the present invention may be 0.7 to 1.5, 0.7 to 1.3, or 0.7 to 1.1, and the UpA value of the ORF7 region of the mutant is 0.3 to 1.0, 0.3 to It may be 0.9 or 0.3 to 0.8, but is not limited thereto.

In one example, the ORF7 sequence of the PRRSV mutant provided by the present invention may include a silent mutation at a position after base 177 from the 5' end of the ORF7 region consisting of the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5. The silent mutation may be freely introduced within the desired range for lowering the CPB of the ORF7 region, for example, 7 to 30, or 7 to 22 bases may be substituted. If fewer than 7 silent mutations are introduced, the virus may not be sufficiently attenuated, and if the number of silent mutations is too large, the virus may not be generated. If the number of silent mutations is greater than the upper limit, virus viability may become impossible due to excessive attenuation.

In one example, the ORF7 region of the PRRSV mutant provided by the present invention may consist of the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 10.

In one embodiment, the ORF7 region of the PRRSV mutant provided by the present invention is the ORF7 region consisting of the nucleic acid sequence of SEQ ID NO: 2, wherein C at position 123 is substituted with T, A at position 129 is substituted with C, and T at position 177 is C substitution, 180th T is substituted with C, 186th T is substituted with C, 189th C is substituted with T, 204th C is substituted with T, 213th T is substituted with G, 222th C is substituted with G, 264th A is substituted with C, 267th A is substituted with T, 288th T is substituted with C, 291th G is substituted with T, 297th C is substituted with T, 309th T is substituted with C, 315th A is substituted with C C substituted with T, 327th T with C, 330th T with G, 336th A with G, 339th G with A, 348th T with A, and 354th G may include mutations consisting of 7 or more, 14 or more, 16 or more, 18 or more, or 22 or more mutations selected from the group consisting of mutations substituted with T.

The parent strain of the PRRSV mutant may be a European porcine reproductive and respiratory syndrome virus, and in one example, may be an E38 strain. The PRRSV mutant strain provided by the present invention may be classified as a European porcine reproductive and respiratory syndrome virus (type 1).

The attenuated PRRSV mutant provided by the present invention has a TCID ₅₀ value of 0.99 times or less, 0.95 times or less, 0.7 times or less, 0.6 times or less, or 0.5 times or less when infected with lung macrophages compared to the parent strain. It is not limited. _{In one embodiment, as a result of measuring the TCID 50} value by infecting lung macrophages with the E38 virus and the mutant strain introducing the virus into the lung macrophages, all mutants showed a lower TCID ₅₀ value than the parent strain, Low proliferation was confirmed. In one embodiment, the virus mutant line having the ORF7 sequence consisting of the nucleic acid sequence of SEQ ID NO: 10, when inoculated into lung macrophages (PAM), TCID ₅₀ / mL value at a level of about 65% compared to the parent strain on day 1 after inoculation , and on the 4th day after inoculation, a TCID ₅₀ /mL value of about 35% was shown, confirming significantly lower toxicity compared to the parent strain.

The attenuated PRRSV mutant provided by the present invention has a blood viral titer of 95% or less, 90% or less, 80% or less, 70% or less compared to the titer (Log (genomic copies/ml)) of the wild-type strain when infected with pigs. % or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, or 35% or less. In one embodiment, as a result of infecting pigs with the E38 virus and the mutant strain introducing the mutation, all of the mutants showed lower viral titers than the parent strain, resulting in a low maximum titer for propagation in the pig and rapidly disappearing. It was confirmed that a safe strain was generated.

The present invention also provides a method of making an attenuated porcine reproductive and respiratory syndrome virus (PRRSV).

The method for producing attenuated PRRSV provided by the present invention comprises the steps of introducing a silent mutation at a position after base 177 from the 5' end of the ORF7 region in the genome of wild-type PRRSV; and generating a virus (mutant strain) into which the mutation is introduced.

The 5' end of the ORF7 region refers to a region corresponding to the 5' end of the polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 2 among the PRRSV genome nucleic acid sequences. When the silent mutation is introduced at a position other than ORF7, a problem in that virus production is not achieved may occur.

The silent mutation introduced into the mutant may lower the CPB value of the ORF7 region of the mutant compared to the ORF7 region of the wild-type virus. The description of the CPB is the same as described above.

The position, number, and type of the silent mutation are the same as the silent mutation introduced into the virus mutant.

The wild-type PRRSV may be a European-type (Type 1) porcine reproductive and respiratory syndrome virus, and in one example, may be strain E38.

In the step of introducing the silent mutation, a person skilled in the art can select and use an appropriate method without limitation within the scope of the purpose for introducing the point mutation, for example, synthesis of a base sequence, use of a primer pair into which the mutation is introduced, And/or a method using a commercially available kit may be used, but is not limited thereto.

In one embodiment, the infectious clone of the PRRSV virus mutant into which the mutation is introduced has a plurality of virus genomes, for example, 2 to 10, 2 to 8, 3 to 10, 3 to 8, 5 to 10. synthesizing DNA corresponding to each fragment by dividing it into 5 or 8 fragments; and linking each fragment to prepare an infectious clone of a virus mutant strain. In one embodiment, the infectious clone of the PRRSV virus mutant into which the mutation is introduced, the virus divides the genome into 6 fragments to synthesize DNA corresponding to each fragment, and then connects each fragment to prepare an infectious clone of the virus mutant. .

In one example, the step of linking each fragment may include treating each of the prepared fragments with a restriction enzyme and linking with a ligase.

The present invention also provides a pharmaceutical composition for preventing or treating porcine reproductive and respiratory syndrome, comprising the attenuated PRRSV mutant and/or the subcultured progeny of the mutant.

The contents of the PRRSV mutant are the same as described above.

The porcine genital and respiratory syndrome may be caused by a European (type 1) porcine genital and respiratory syndrome virus.

The passage cultured progeny are passages 1 to 80, passages 1 to 70, passages 1 to 60, passages 1 to 50, passages 1 to 40, passages 1 to 30, passages 1 to 20, or passages 1 to 10. It may include a cultured progeny virus.

The composition for preventing or treating porcine reproductive and respiratory syndrome of the present invention may include additional components known to those skilled in the art, and may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

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, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient in the composition, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. is prepared In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. may be used.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the individual, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. For example, for a desirable effect, the composition of the present invention may be administered in an amount of 0.0001 to 1,000 mg/kg (body weight) per day. The administration of the composition may be administered once a day or divided into several administrations.

In one embodiment, the pharmaceutical composition may be a vaccine composition. The vaccine may be a live vaccine and/or a dead vaccine, and specifically, the attenuated PRRS chimeric virus described herein may be a live modified vaccine containing one or more of the aforementioned strains in a live state in a pharmaceutically acceptable carrier. . Additionally, or alternatively, the inactivated virus may also be used to prepare a dead vaccine.

The vaccine may include a PRRSV mutant at an appropriate concentration in consideration of the body weight, age, dietary stage, and/or immunity of the subject to be administered in the range for the prevention of PRRS. For example, the dose of the virus mutant in the vaccine composition is in the range of TCID ₅₀ 2 to 6, or TCID ₅₀ 3 to 4, but may vary depending on the type of individual, but is not limited thereto.

The vaccine composition of the present invention may be administered to pigs, and the pigs may be pigs in one or more growth stages selected from the group consisting of weaning piglets, growing stages and finishing stages. The pig of the weaning piggy stage means a pig from 7 days or more, 14 days or more, or 21 days or more to before reaching a weight of 30 kg, the growing stage is a period when the pig's weight is 30 to 50 kg, the finisher stage It may mean the period after the upbringing period. The pig may be a pig and / or a wild boar, regardless of breed, for example, at least one selected from the group consisting of Landrace, Yorkshire, Duroc, Berkshire, and Korean native pigs, 2 It may be more than one species, three or more, four or more, or five or more, and includes all pigs born by crossbreeding between the species.

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

The attenuated PRRSV mutant of the present invention, or a vaccine composition comprising the same, is administered via oral, parenteral, subcutaneous, intramuscular, intradermal, sublingual, transdermal, rectal, transmucosal, surface area via inhalation, buccal administration, or a combination thereof. can be administered. In addition, the attenuated PRRSV mutant may be administered in the form of an implant capable of allowing sustained release of the attenuated virus.

The attenuated PRRSV mutant or vaccine composition comprising the same of the present invention may be administered via injection, inhalation or transplantation, but is not limited thereto. Depending on the desired duration and effectiveness of vaccination or treatment, the attenuated PRRSV mutant or vaccine composition comprising the same may be administered once or several times, and also intermittently, for example, in the same amount or in different doses daily for several days, weeks or months. can Injections may be given by injection in the desired amount or by subcutaneous or nasal spray, or alternatively, continuous infusion.

In one embodiment, the pharmaceutical composition may be provided in the form of a kit for preventing or treating porcine reproductive and respiratory syndrome. Said kit comprises a container, preferably a vaccine composition containing the attenuated PRRS chimeric virus of the present invention, a pharmaceutically acceptable carrier, adjuvant and clinical signs or effects of PRRS infection, preferably alleviating the frequency or severity of PRRS. Instructions for administering the immunogenic composition to an animal in need thereof may be included. The kit may also include means for injection and/or other forms of means of administration. The kit may also include a solvent. The attenuated vaccine may be lyophilized and reconstituted with a solvent to provide a solution for injection and/or inhalation. The solvent may be water, physiological saline, a buffer or an adjuvant solvent. The kit may comprise a separate container containing the attenuated virus, a solvent and/or a pharmaceutically acceptable carrier. Instructions for use may be labels and/or printed materials affixed to one or more containers.

The mutant strain of the porcine reproductive and respiratory syndrome virus provided by the present invention is attenuated than the parent strain, so it has low pathogenicity, high stability, and can be used as a vaccine for effective prevention and/or treatment of PRRS disease by improving the immunity of pigs.

1 is a result of aligning and comparing the nucleic acid sequences of the E38 parent strain and the ORF7 region of each mutant strain. The color of the circle in front of the SEQ ID NO: legend indicates the success or failure of generation of a virus containing ORF7 of each nucleic acid sequence (blue: successful production, red: production failure).
2 is a graph showing the results of confirming the proliferation of the virus over time after infecting each of the generated viruses with pig lung macrophages (PAM).
3 is a graph showing the viral titer in blood over time after inoculation of each generated virus into pigs. * indicates statistically significant results.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

In the Examples below, since RNA, which is the genome of PRRSV, is easily destroyed, all PRRSV genome treatments were performed by synthesizing DNA, and then RNA was prepared therefrom and used for transfection of cells unless otherwise stated.

Preparation Example 1. Information on the parent strain

As the parent strain for preparing the mutant strain, the E38 strain, which is a European PRRSV isolated in Korea, was provided by the Quarantine Headquarters, and the entire genome sequence of the E38 strain is shown in SEQ ID NO: 1.

The results of comparing the homology of the entire genome sequence between the domestic isolate E38 and the American PRRSV (VR-2332) and European PRRSV (Amervac, Lelystad and Lena strains) strains are shown in Table 1 below.

PRRSV type North American
(Type II) European
(Type I) virus strain VR-2332 Amervac Lelystad Lena Sequence homology with E38
(Identity, %) 58.2 88 88.9 79.2

Preparation Example 2. Codon pair deoptimization

Using the E38 virus as a parent strain, an attenuated infectious clone was prepared by applying codon pair deoptimization in the genome synthesis step.

NSP1 and ORF-7 were selected as the genes to be subjected to codon pair deoptimization, respectively, using the generally known SAVE (Synthetic Attenuated Virus Engineering) program or the SAVE (Synthetic Attenuated Virus Engineering) program developed by the present inventors. It was prepared by silent mutation of some of the nucleotide sequences according to the principle of codon pair deoptimization.

First, using the SAVE program, the CPB (Codon Pair Bias) value, which is a bias caused by the interaction of gene codons of E38 (domestic isolate) virus when they are arranged in pairs, was quantified using a computer algorithm. The CPB level fluctuates when some nucleotide sequences of the viral gene are substituted with other nucleotides, and is known to be closely related to the proliferation of the virus. When the CPB level is decreased through nucleotide sequence substitution (Deoptimization), the proliferation of the virus is reduced and attenuated.

Comparative Example 1. Preparation of codon de-optimized virus mutants for the NSP1 region

1-1. Codon pair deoptimization

The gene region encoding the NSP1 protein in PRRSV is known to have high genetic stability in the genome of PRRSV. Therefore, using the SAVE program for the NSP1 gene region of E38 (domestic isolate) PRRSV, codon pair deoptimization was applied to the base region with a relatively high CPB value.

CPB, CpG and UpA values before and after codon pair deoptimization for each strain designed by the above method are shown in Table 2 below. The CpG and UpA levels are inevitably generated in the codon pair deoptimization process, and it is known that when the CpG or UpA levels in the gene increase, cell stress is induced and the proliferation of the virus is reduced.

parent strain Codon Deoptimization CPB CpG UpA nt E38 X (original) -0.0511 0.7361 0.4878 - E38 O (NSP1) -0.2491 1.0859 0.8464 78

As can be seen in Table 2, as a result of applying codon deoptimization to the NSP1 gene, the CBP value decreased in both the E38 and Lelystad virus strains, and the CpG and UpA levels also increased, so that the virus having the above mutated genome It was suggested that the proliferative property of the strain was decreased compared to the parent strain.

1-2. Confirmation of production of infectious strains of virus mutants

In order to generate the designed mutant strain, the genome of the E38 or Lelystad strain (including codon de-optimized mutations) was divided into 6 fragments to synthesize DNA, respectively. Each fragment was treated with restriction enzymes and then ligated to prepare one infectious clone.

The infectious clone was inserted into a high copy vector and then transfected into BHK cells (Korea Cell Line Bank) using lipofectamine to attempt to generate an infectious virus.

However, the generation of the E38 virus mutant with codon deoptimization of the NSP1 gene was not made.

Example 1. Preparation of codon deoptimization virus mutants for the ORF7 region

1-1. Codon pair deoptimization in the ORF7 region

ORF7 is a region present at the 3' end of the PRRSV genome, and refers to a region corresponding to a length of 387 nt of the 3' end of the genome .

In substantially the same manner as in Comparative Example 1-1, codon pair deoptimization for the ORF7 site for the E38 virus was performed using the SAVE program, and thus a mutant in which a silent mutation was introduced into the ORF7 site was designed. . Table 3 below shows the CPB, CpG, UpA and nt values of the parent strain and the mutant strain. Description of each numerical value is the same as described in Comparative Example 1-1.

Codon Deoptimization CPB CpG UpA nt X (parent strain) -0.0371 0.5714 0.2610 - O (ORF7, 7) -0.0963 0.7520 0.3589 7 O (ORF7, 22) -0.2251 1.0797 0.6185 22

In Table 3, the ORF sequences of the parent strain and each mutant are shown in SEQ ID NOs: 2 to 4. Specifically, SEQ ID NO: 2 is the ORF7 region nucleic acid sequence of the parent strain, SEQ ID NO: 3 is the ORF7 region nucleic acid sequence of the strain containing 7 mutations, and SEQ ID NO: 4 is the strain containing 22 mutations in Table 3 above. ORF7 site nucleic acid sequence.

The nucleic acid sequence of SEQ ID NO: 3 is, in the nucleic acid sequence of SEQ ID NO: 2, 177th T to C, 180th T to C, 204th C to T, 213th T to G, and 222th C to G , in which A at position 264 is replaced by C, and A at position 267 by T, respectively.

The nucleic acid sequence of SEQ ID NO: 4 is, in the nucleic acid sequence of SEQ ID NO: 2, 48th A to G, 108th C to G, 111th G to A, 123rd C to T, and 129th a to c , 153th T to C, 162th A to G, 171st C to G, 174th G to A, 177th T to C, 180th T to C, 186th T to C, 189th C to T, 204th C to T, 213th T to G, 222th C to G, 264th A to C, 267th A to T, 285th C to T, 288 Silent point mutations in which the th T is substituted with C, the 291st G with T, and the 297th C with T, respectively. That is, the nucleic acid sequence of SEQ ID NO: 4 includes all of the point mutations of SEQ ID NO: 3, while 48, 108, 111, 123, 129, 153, 162, 171, 174, 186 and further 15 mutations at positions 189, 285, 288, 291 and 297.

In the case of the mutant strain using E38 as the parent strain, the CPB level decreased and the CpG and UpA levels increased as the number of mutations introduced into ORF7 increased.

1-2. Generation of infectious strains of virus mutants

Each virus mutant (codon deoptimized virus mutant) designed in Example 1-1 was generated in substantially the same manner as in Comparative Example 1-2.

As a result, the generation was successful only in the case of a mutant having ORF7 consisting of the nucleotide sequence of SEQ ID NO: 3, in which silent mutations were introduced into 7 bases, whereas 22 silent mutations were introduced into ORF7 of E38, SEQ ID NO: 4 The generation of a mutant strain having ORF7 consisting of a nucleotide sequence was not performed.

Example 2. Virus generation according to the number and location of silent mutations in ORF7

2-1. Confirmation of virus production according to the number of silent mutations

Based on the virus production results in Example 1-2, in order to determine whether the virus is generated dependent on the number of silent mutations for the E38 parent strain, 14, 16, Silent mutations in which 18 and 20 bases were substituted were introduced to confirm whether or not the virus was generated.

Specifically, the ORF7 base sequence of the mutant strain (E38_ORF7_CPD14_NUM_O) in which 14 bases of ORF7 are substituted (E38_ORF7_CPD14_NUM_O) is SEQ ID NO: 5 and the ORF7 base sequence of the mutant strain (E38_ORF7_CPD16_NUM_X) in which 16 bases are substituted is SEQ ID NO: 6, the mutant in which 18 bases are substituted The ORF7 base sequence of (E38_ORF7_CPD18_NUM_X) is SEQ ID NO: 7, and the ORF7 base sequence of the mutant strain (E38_ORF7_CPD20_NUM_X) in which 20 bases are substituted is shown in SEQ ID NO: 8.

More specifically, the nucleic acid sequence of SEQ ID NO: 5 is the 123th C to T, the 129th A to C, and the 177th T to C in the ORF7 nucleic acid sequence of the parent strain (E38) represented by the nucleic acid sequence of SEQ ID NO:2. , 180th T to C, 186th T to C, 189th C to T, 204th C to T, 213th T to G, 222th C to G, 264th A to C, contains a silent mutation in which A at position 267 is replaced by T, T at position 288 by C, G at position 291, and C at position 297 by T.

The nucleic acid sequence of SEQ ID NO: 6 includes all of the mutations of SEQ ID NO: 5, and further includes a mutation in which the 108th C is substituted with G and the 111th G is substituted with A.

The nucleic acid sequence of SEQ ID NO: 7 includes all of the mutations of SEQ ID NO: 5, and additionally, the 153rd T is substituted with C, the 162th A is substituted with G, the 171st C is substituted with G, and the 174th G is substituted with G Further includes a mutation substituted with A.

The nucleic acid sequence of SEQ ID NO: 8 includes all mutations of the nucleic acid sequence of SEQ ID NO: 6 and SEQ ID NO: 7. Specifically, it includes all the mutations of SEQ ID NO: 5, and additionally, the 108th C to G, the 111th G to A, the 153th T to C, the 162th A to G, and the 171st C to G , and a mutation in which the 174th G is substituted with A.

Virus generation was performed in substantially the same manner as in Comparative Example 1-2, and only the E38_ORF7_cpd14_num_o mutant succeeded in virus generation, but the remaining three mutants (E38_ORF7_cpd16_num_x, E38_ORF7_cpd18_num_x and E38_ORF7_cpd20_num_x) failed to generate.

Therefore, when there were more than 14 silent mutations, it was confirmed that factors other than the number of silent mutations were involved in virus generation.

2-2. Virus generation according to silent mutation location

Based on the results of Example 2-1, in order to determine whether the location of the silent mutation affects the production potential of the virus, a virus having 22 mutations not generated in Example 1-2 and the above Examples Based on the E38_ORF7_cpd18_num_x used in 2-1 and the mutation in the common position, 18 silent mutations (E38_ORF7_cpd18_position_O) and 22 (E38_ORF7_cpd22_position_O) were introduced in the ORF7 base sequence (SEQ ID NO: 2) of the parent strain, respectively. and virus production was confirmed.

The E38_ORF7_cpd20_position_O mutant has an ORF7 region consisting of the nucleic acid sequence of SEQ ID NO: 9, and specifically, in the wild-type ORF7 region consisting of the nucleic acid sequence of SEQ ID NO: 2, C at position 123 is substituted with T, and A at position 129 is replaced with C Substitution, T at position 177 is substituted with C, T at position 180 is substituted with C, T at position 186 is substituted with C, C at position 189 is substituted with T, C at position 204 is T Substitution, T at position 213 is substituted with G, C at position 222 is substituted with G, A at position 264 is substituted with C, A at position 267 is substituted with T, T at position 288 is substituted with C Substitution, G at position 291 is substituted with T, C at position 297 is substituted with T, T at position 309 is substituted with C, C at position 315 is substituted with T, T at position 327 is substituted with C substitutions, and mutations in which T at position 330 is substituted with G.

The E38_ORF7_cpd22_position_O mutant line has an orf7 sequence consisting of the nucleic acid sequence of SEQ ID NO: 10, and specifically includes all mutations of the E38_ORF7_cpd18_position_O strain among the orF7 regions of the wild-type virus consisting of the nucleic acid sequence of SEQ ID NO: 2, and additionally at position 336 of A is substituted with G, G at position 339 is substituted with A, T at position 348 is substituted with A, and G at position 354 is substituted with T.

The generation of each virus was performed in substantially the same manner as in Comparative Example 1-2, and both strains E38_ORF7_cpd18_position_O and E38_ORF7_cpd22_position_O were successfully generated.

1 shows the results of sequence alignment centered on the mutation site of the ORF7 position of each virus designed in Examples 1-1 to 2-2. Unlike the E38_ORF7_CPD14_NUM_O mutant (ORF7 nucleic acid sequence: SEQ ID NO: 5), which succeeded in generating a virus, only bases 108 and 111 in the ORF7 sequence of SEQ ID NO: 5 were further substituted (E38_ORF7_cpd16_num_X, ORF7 nucleic acid sequence: SEQ ID NO: 6) and the mutant strain (E38_ORF7_CPD18_NUM_X) in which the 153rd, 162th, 171st, and 173th bases of the ORF sequence of SEQ ID NO: 5 were substituted. E38_ORF7_cpd20_num_x containing both mutation positions of the two mutants of E38_ORF7_cpd16_num_X and E38_ORF7_cpd18_num_X and a virus mutant containing an ORF7 site consisting of the nucleic acid sequence of SEQ ID NO: 4 also did not generate a virus, so the nucleic acid of SEQ ID NO: 5, which is the position where each mutation was introduced It was confirmed that base 48, bases 108 to 111, or bases 153 to 173 in the sequence should be excluded from codon pair deoptimization.

After that, the strain (18 base substitution strain, e38_orf7_cpd18_position_o) in which the bases 309, 315, 327, and 330 behind the base 297 were additionally substituted to the strain 14 base substitution (E38_ORF7_cpd14_num_O) were successfully generated, and the 18 base substitution strain A strain substituted with bases 336, 339, 348, and 358 in which 4 bases were substituted in the ORF7 sequence (22 base substituted strain, e38_orf7_cpd22_position_o) was also successfully generated.

For codon pair deoptimization to obtain an infectious clone of the attenuated European type (Type I) PRRSV through the above data, a substitution site must be applied after base 177 of the ORF7 site consisting of the nucleic acid sequence of SEQ ID NO: 2 was confirmed.

Example 3. Confirmation of attenuation of virus mutants

Attenuation was confirmed in vivo for the four successful viruses (E38_ORF7_cpd7_O, E38_ORF7_cpd14_num_o, E38_ORF7_cpd18_position_o, and E38_ORF7_cpd22_position_o). As a control, E38 (original), the parent strain of the mutants, was used.

After infecting pig lung macrophages (Porcine Alveolar Macrophage, PAM) with the above four mutants or parent strains, respectively, the proliferation of each virus was confirmed _{by TCID 50 measurement.}

More specifically, PAM cells per well in a ^6- well plate containing 2 ml of RPMI medium (including 10% (v/v) FBS, 1% (w/v) penicillin, and streptomycin) at a ratio of 2 X 10 6 cells/well was busy. Next, the generated strain and the parent strain were inoculated into different wells at a ratio of MOI 0.01, and after removing all the supernatant 1 hour after inoculation, 2 ml of the maintenance solution RPMI medium (composition homologous) was dispensed. Thereafter, the supernatant of each well was collected every 1, 2, 3, and 4 days, and TCID ₅₀ (Tissue culture infective dose 50) was measured. For TCID _50, ^{2 X 10 5} cells/well of MARC-145 cells were aliquoted together with 100ul of DMEM medium (10%(v/v) FBS, 1%(w/v) penicillin, streptomycin) in a 96-well plate the day before the test. attached was used.

200ul of the collected supernatant was dispensed in a row to the far left well of a 96-well plate, and 180ul of DMEM medium (FBS, antibiotic-free) was dispensed into the remaining wells. Next, 20ul each was collected with a multi-pipet in turn from the left and diluted by decimal while dispensing into the right well to prepare the virus for inoculation. The tips were changed and diluted sequentially, and the last well of the 12th well was maintained as a negative control (DMEM medium without supernatant).

The prepared virus dilution was inoculated on a plate to which MARC-145 cells were attached. At this time, all the culture medium of MARC-145 cells was removed, and 200ul of PBS was dispensed into each well and discarded 3 times to perform a washing process. Then, the prepared virus diluted solution was dispensed and inoculated by 100ul per well, and after 2 hours of inoculation, all the inoculation solution was removed and a new maintenance solution (DMEM medium, 10%(v/v) FBS, 1%(w/v) penicillin, streptomycin) ) was dispensed 100ul. It was checked whether clumping and apoptosis, a type of cytopathogenic effect (CPE), occurred in the cells for about 7 days after dispensing.

The final value is based on the previous dilution factor of the well in which no CPE phenomenon was observed, and the dilution factor at which 4 wells (half of the 8 virus inoculum) can show CPE was obtained.

2 shows a graph of the TCID _{50 analysis result.} Data marked with * in FIG. 2 means that there was a statistically significant difference, and all strains were expressed in the form of 'E38-number of mutations' for convenience. All four mutants showed lower proliferation compared to the parent strain (E38-original), and the strains with a lot of codon pair deoptimization were attenuated, and in particular, the E38-18 and E38-22 strains were statistically significantly attenuated. confirmed that it was.

Specifically, in the case of E38-22 strain it showed about 65% of the TCID ₅₀ / mL value compared to the parent strain on day 1 after challenge, day 4 after inoculation is approximately 35% of the indicated TCID ₅₀ / mL value of the parent It was confirmed that it was significantly attenuated compared to the strain.

Example 4. Analysis of Proliferative (Titer) in Pigs of Viral Mutants

For the analysis of the proliferation in the pig body of each virus mutant generated, each virus was inoculated into experimental pigs, and then the RNA content of the virus in the blood was measured using real-time PCR. Information on the pig groups used in each experiment is shown in Table 4 below.

group inoculated strain Marisu T01 (control) E38 (Original) 5 T02 E38_ORF7_cpd7_o 5 T03 E38_ORF7_cpd14_num_o 5 T04 E38_ORF7_cpd18_position_o 5 T05 E38_ORF7_cpd22_position_o 5

Pigs used in the experiment were SPF pigs, and 3-week-old piglets that were confirmed not to show any antigen-antibody reaction to PRRSV were used. Next, weaned 3-week-old piglets were inoculated with _{each virus in the nasal cavity by 50 /2ml of 5 TCID.} On days 0 (on the day of inoculation), 7, 14, 21, and 28 from the day of inoculation, 3 ml of blood was collected from the jugular vein of pigs, serum was separated, and the virus RNA contained in the isolated serum was analyzed using Intron's Viral gene-spin. It was extracted using a viral DNA/RNA extraction kit.

Specifically, 150ul of the collected serum was mixed with 250ul of lysis buffer, vortexed for 15 seconds, incubated for 10 minutes at room temperature, and 350ul of binding buffer was mixed and then lightly vortexed. A total of 750ul mix was transferred to the column and centrifuged at 13,000 rpm for 1 minute, the filtrate was discarded, 500ul of washing buffer A was added to the column, and centrifuged at 13000rpm for 1 minute in the same manner. The filtrate was discarded again, and 500ul of washing buffer B was added to the column, followed by centrifugation at 13000rpm for 1 minute. The last filtrate was discarded and centrifuged again in the same way to remove all alcohol components. Elution buffer 30ul was added, incubated for 1 minute, and finally RNA was extracted by centrifugation.

2ul of the extracted RNA was reverse transcribed using TOPscript RT DRY MIX kit (Enzynomics) to synthesize cDNA. The reaction was terminated by mixing 2ul of RNA and 18ul of DEPC DW extracted into the tube of the kit already prepared in a dry state, incubating at 50℃ for 1 hour, and then incubating at 95℃ for 10 minutes.

Next, the amount of synthesized cDNA genomic copy detected per 1ml of serum was measured using the Realtime method. Sybr green mix (thermos fisher) 10ul of DEPC DW and 7.5ul of PRRSV specific primer set (SEQ ID NOs: 11 to 12) shown in Table 5 was mixed with 0.5ul of 10 pmol of the cDNA synthesized above into a mix of 2ul and PCR reaction was performed. did. Specifically, DNA was amplified by annealing at 95° C. for 30 seconds, annealing at 60° C. and elongation at 60° C. for 1 minute, and a reaction to obtain a dissociation curve was additionally performed. A total of 35 cycles of reaction were repeated, and the Ct value was calculated for each sample according to the threshold value, and 33 or more were excluded. The Ct value was converted to a genomic copy by substituting it into a standard curve and expressed.

PRRSV-specific primers base sequence (5'>3') SEQ ID NO: Forward primer (PRRSV_forward) TGGCCAGTCAGTCAATCAAC 11 Reverse primer (PRRSV_reverse) AATCGATTGCAAGCAGAGGGAA 12

Fig. 3 shows a graph of the virus titer in blood according to the lapse of time after virus inoculation of each group. In FIG. 3, * indicates a statistically significant result, and each virus is indicated in the form of 'E38-number of mutations' for convenience of description. As can be seen in FIG. 3 , the blood virus titer of pigs measured 7 days, 14 days, and 21 days after virus inoculation was statistically significantly lower than that of the existing parent strain E38 when E38_ORF7_cpd22_position_o was inoculated. After inoculation, it continuously maintained a low titer of 33% to 63% compared to that of the parent strain, and on the 28th day, the titer of E38_ORF7_cpd22_position_o was measured as 0, confirming that the proliferative potential in the pig body was very low.

Through this, it was confirmed that through codon pair deoptimization for the European PRRSV of the present invention, a safe strain with a low maximum titer that can proliferate in vivo and rapidly disappearing can be made.

Korea Institute of Bioscience and Biotechnology KCTC14016BP 20191105

<110> BioPoA, Inc. REPUBLIC OF KOREA(Animal and Plant Quarantine Agency) <120> Method for producing European porcine reproductive and respiratory syndrome virus and use thereof <130> DPP20200149KR <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 15044 <212> DNA <213> Artificial Sequence <220> <223> E38 full genome sequence <400> 1 atgatgtgta gggtattttc cctacgtgcg caacactctt agtgtttgtg taccttggag 60 gcgtgggtat agccccgccc caccccttgg cccctgttct agcccaacag gtatccttct 120 ctctcggggc gagtgcgccg cctgctgctc ccttgcagcg ggaaggacct cccgagtatt 180 tccggagagc acctgcttta cgggatctcc accctttaac catgtctggg acgttctccc 240 ggtgcatgtg caccccggct gcccgggtat tttggagcgc cggccaagtc ttttgcacac 300 ggtgtctcag tgcacggcct cttctccctc cagagctcca ggaccccggt cttggcgcaa 360 ttggcttgtt ttacaagccc aaagacaagc tccattggaa agtccctgtc ggcatccctc 420 aggtagaatg cactccatct gggtgctgtt ggctctcagc tatttttccc ttggcacgta 480 tgacttccgg caaccacaac tttctccaac gacttgtgaa ggttgctgat gttttgtacc 540 gtgacggttg cttggcacct cgacatcttc gtgaactcca ggtttacgag cgcggttgta 600 actggtaccc gatcacgggg cccgtgcccg ggatgggttt gtttgcgaac tccatgcatg 660 tatctgacca accattccct ggtgccactc atgtgctgac taactcgcct ctgcctcaac 720 aggcttgtcg gcagccgttc tgtccatttg aggaggctca ttctaacgtg tttaggtgga 780 acaaattcgt ggttttcatg gaccccccct ccggcggtag atgccgcatg atgtggacac 840 ctggatccga cgactcgact aacttggggg cgttgccgcc tgacctggaa cgtcaagtcg 900 agattctcac ccggagcttt cctgcttacc atcctgtcaa cctcagcgac tgggaactca 960 ccgagtcccc tgaacatggt ttttccttca gcacgtctca ttcttgtggt taccttgccc 1020 aaaaccctga tgggtttgat ggtaagtgtt ggctttcctg ttttcttgat ctgtcaaccg 1080 aagtgtggcg tcatgaggag tatctagctg acaccctcgg ttatcaaacc aagtggggcg 1140 tacatggcaa atatctccag cgtaggcttc aaatcaatgg tgtccgtgct gtggttgatc 1200 cagatggtcc cattcacgtt gaagcgctgt cttgccccca gtcttggatc aggcacctga 1260 ctctggacga tgaagtgacc ccagggttca ttcgcctgac gtctctccgc atcgtgccga 1320 atacggagcc taccaccctc cgggtctttc gatttgggac gcataagtgg tatggcgctg 1380 ccggtaaacg agctcgcgct aagcgtgccg ccaaaagcga gaagaactcg gctactgccc 1440 ccaaggctac cgaaccaatt cctgcctgtg aaactaccat ctattccccg ccaacagacg 1500 gatcttgtgg ttggcatgtt cttgccgcca taataaaccg gatggtgaat gatgatttca 1560 cgtcccctct gacccagtac aacagaccag aggacgattg ggcatctgat tgcgaccttg 1620 ctcaagcgat tcaatgcctg caactacccg ctactatagt ccggagtcgt gcctgtccca 1680 atgctaaata cctcataagg cttaacggtg ttcactggga agtggaggtg aggcctggga 1740 tggcccctcg ctcccttcct cgcgaatgtg tggttggcgt ttgttccgaa ggctgtgtcg 1800 cgtcgtctta cccagagaat gggctacctg aacgagcgct tgaagccttg gcgtctgctt 1860 acagactgcc ttccgactgc gtcagttccg gtattgctga ctttcttgct gacccccctc 1920 aggagttctg gactcttgac aaaatgctga cttccccgtc accagagcaa tctagctttt 1980 ctagtttgta caaattgcta ttagaggttg ttccgcaaaa atgcggtgcc acggaggggg 2040 ctttcatcta tgccgttgat aggatgttaa atgattgtcc gagttctaag caggccatgg 2100 cactcttagc aaaaattaag gttccatcct caaaggcccc atctgtgtcc ttagacgagt 2160 gttttcctac gaatgttccg gccgacctcg agctagcacc tcagaagaag ccccaaagtt 2220 ccggcactgt tgatgtcctg cgttcaccgg gcgcaaaaga gttcgaggaa gcagccctag 2280 aagaagtcca agagggtggc tacaagactg tccattctgt accccatacc aagggtccta 2340 ataaagagca ggtacaagtg gttgtcggtg aacagctgaa gctcggcggt tgtgatttgg 2400 tagtcgggaa tgctcacaaa gatgtcctgg tctcagccgg tccaattgac ttagcaagcg 2460 aaccgctgga tctgtcccga ccagcaccag ctgccacaac gacccctgag ggagaacaca 2520 cacccgaaaa tccaggttct gatgctggtg ccctccttgt taccactcga aagttcgtct 2580 cagcggggct tatgctccat catgttgagc actgcggtac ggagtcgggc gacggcggtt 2640 cgcctttgga tctgtctgat gcgcaaatcc cggaccagcc tttagacctg tccctagctg 2700 cctggccagt gaagtccacc gcgtctgatc ctggctgggt tcacggtagg cgcgaaccag 2760 tctttgtaaa accccgaaat gttttctcgg atggcgattc ggtccttcag tttggggggc 2820 tttctgaatc cagctctgtc atcgagtttg accggacaaa agacaccccg gtggtcgacg 2880 gccctgtcga cttgacgact tcgaacgagg ccctctctat ggtcgatcct cttgaatttg 2940 ccgaactcaa acgcccgcgt ttttccgcac aagccttaat tgaccgaggc ggtccacttg 3000 ctgacgttca tgcgaagata aaaaatcaag tgtatgaacg gtgccttcaa gcctgtgagc 3060 ccggcagtcg tgcaacccca gccaccaaga agtggcttga cagaatgtgg gctagggtgg 3120 atatgaaaac ttggcgctgt acctcacagt tccaagccgg tcgcattctt gcgtccctca 3180 aattcctccc tgacatgatt caagacacac cgcctcctgt tcccaggaag aaccacgcca 3240 gtgacagtgc cggcttgaag cgactggtag cacagtggga tagaaaatta agtgtgacct 3300 cccttcaaag gccggttgaa ctagtgcctg accagattgt cccctcgcct ataggcgtcc 3360 agcaagatgg cgttactccc tccaatgggc caccccaagc gccggatctt cctggccggg 3420 tggacgcggg cggaagttgg aaaggcctca tgctttctgg cacccgtctt gcggggtcca 3480 tcagtcagcg cctcatgaca tgggtttttg aaatcctttc ccatctccca gcttttatgc 3540 tcacactttt ctcgccacgg ggctctatgg ctccaggtga ttggttgttt gcaggtgttg 3600 ttttacttgt tctcctgcta tgtcgttctt acccaatact cgggtgccta cccttattgg 3660 gtgtcttttc tggttctgtg cggcgcgttc gtctgggtgt ttttggttct tggatggctt 3720 ttgctgtatt tttattctca actccaaccg gcccagtcgg ttcttcttgt gaccacgatt 3780 cgccggagtg tcatgctgag cttttggctc ttgagcagcg ccaactttgg gaacctgtgc 3840 gcggtcttgt ggtcggcccc tctgggctcc tatgtgtcat tcttggtaag ttactcggtg 3900 ggtcacgtta tctctggcat attttcctac gtttatgcat gcttgcggat ctgtcccttt 3960 ctcttattta tgtggtgtcc caggggcgtt gtcacaagtg ttggggaaag tgtgtaagaa 4020 cggctcctgc agaggtggct ctcaatgtat ttccttttac gcgcgccact cgttcttctc 4080 ttgtgaactt gtgtgaccgg ttccaagcgc caacaggggt tgaccccgtg tacttggcga 4140 caggttggcg cgggtgctgg cgcggcgaaa gtcccatcca ccaatcacac caaaaaccca 4200 tagcttatgc caacttggat gaaaagaaaa tatcagccca aacggtggtt gctgtcccgt 4260 atgatcctag tcaggccatc aaatgtctga aagttctgca ggctggaggg gccatcgtgg 4320 atcagcccgt acctgaggtc actcgtgtgt ccgaaatccc cttctcggcc ccattcttcc 4380 caaaactacc agtcaacccg aattgcagga ttgtggtaga ttcggacact tttgtggctg 4440 cggttcgctg cggttattcg acggcgcaac tggtcctagg tcgtggcaac tttgccaagt 4500 taaaccaggt cccttccagg aactccacct ccaccaaaac gactggtggg gcttcttata 4560 ctcttgctgt ggtccaggtg tccgtgtgga ctctcatcca cttcatcctc ggccttttgt 4620 tgacgtcgcc tcaggtttgt ggccgaggga cctccgaccc gtggtgttct aacccttttt 4680 cgtaccccac ttatggcccc ggtgtcgtgt gctcctctca actttgtgtg tccgccgatg 4740 gtgttaccct gccgttgttc tcagccgtgg cccagctttc tggtagggag gtggggattt 4800 ttatcttggt gctcgtctcc ttggctgctt tggcccatcg catggctctt aaggctgaca 4860 tgctagtggt ctttttggcg ttctgtgctt acgcatggcc catgagctcc tggctaattt 4920 gtttattccc tacactctta aagtgggtca ccctccaccc tctcaccatg ctttggatgc 4980 actcattctt agtgttttgc ctgccggccg ccggcgttct ttcactgggg ataactggtc 5040 tcttctgggc aattggccgc tttacccaag ttgccggact catcacaccc tatgacattc 5100 atcagtacac ctccggaccg cgtggtgcgg ctgctgtggc aacagcccca gaaggtacct 5160 atatggcagc cgtccggcga gctgccttaa ccggacgaac cctgattttc acaccttctg 5220 caattggatc tcttcttgaa ggtgctttta ggtcccacaa gccctgcctt aataccgtga 5280 atgttgtagg ctcttccctt ggttccgggg gagtttttac cattgacggc agaaaaaccg 5340 tggtcactgc cacccatgta ttgaatggcg acgtagctag ggtcaccggc gaatcctaca 5400 accgcatgca cactttcaag actaatggtg attatgcatg gtcccatgct gacgactggc 5460 agggcgttgc ccctgtggtc aaggtctcga aaaggtaccg cggtcgtgct tattggcaaa 5520 catcaaccgg tgtcgagccc ggcattatcg gggaagggtt cgctttctgt tttaccaact 5580 gcggtgactc gggatcacct gttatatctg aagctggtga ccttattggc atccacactg 5640 gttcaaacaa actcggttct ggtcttgtga caacccctga aggggagacc tgctccatca 5700 aagaaactaa actttctgac ctctctaagt actttgcagg tccgagcgtc cctcttgggg 5760 acattaagtt gagcccggcc atcatccctg acgtgacatc tgttccaagt gacttggcat 5820 cacttcttgc ttccgtccct gtgatggagg gcggcctctc aactgtccaa cttttgtgtg 5880 tctttttcct tctctggcgc atgatgggcc acgcctggac acccattgtt gccgtaggtt 5940 tcttcctgct aaatgaaatt ctcccagcag ttttggtccg agccgtgttc tcctttgcac 6000 tctttgttct tgcatgggcc accccctggt ccgcacaggt gttgatgatc agacttctca 6060 cggcggccct caaccgcaac aagctttctt tggtattcta tgctttcgga ggtgttgtcg 6120 gcctagctgc tgaagtcggg acttttgccg gtaggctgtc cgaactgtct caagcccttt 6180 cgacatactg cttcctgcct agagtcctcg ctatgactag ttgtgtcccc atcatcatta 6240 ttggtggact tcatgccctc ggcgtaatct tgtggctatt caaataccgg tgcctccaca 6300 acttgttggt tggcgatgga tgtttttcaa gcgccttctt cctacggtac tttgctgagg 6360 gcaaccttag gaagggtgtt tcacagtcct gtggcatgag caacgaatcc ttgacggctg 6420 ccctagcctg caagttatcg caggctgatc ttgaatttct gtccagcttg acgaacttca 6480 agtgctttgt atctgcttca aatatgaaga atgctgccgg ccagtatatt gaagccgcgt 6540 atgctaaggc cctgcgccaa gagttggcct ctctagttca ggttgacaaa atgaaagggg 6600 ttttggccaa gcttgaggcc ttcgctgaaa cagccacccc gtctcttgac acgggtgacg 6660 tgattgttct gcttgggcaa cacccccacg ggtccgtcct cgacataaat gtggggactg 6720 aaaggaaaac tgtgtccgtg caagagaccc ggagtttagg cggctccaaa ttcagtgttt 6780 gtactgtcgt gcctaacaca cctatggacg ccttaaccga catcccactc cagacaccaa 6840 cccccctttt tgagaatggc ccgcgtcatc gtggcgaaga agacgatctt aaagtcgaga 6900 ggatgaagaa acactgtgta tccctcggct tccacaacat caatggtaaa gtttactgca 6960 aaatttggga caagtccacc ggtgacacct tttatacaga tgattcccgg tacatccaag 7020 accatgcttt tcaggacaga tcagccgact atagggacag ggactatgaa ggtatgcagg 7080 ctgccccccc acaaggattt gatccaaagt ctgaaacccc tgttggtacc gtcgtaatcg 7140 gcggtgtcac gtataacagg tatctagtaa aaggtaaaga ggttctggtt cccaagcccg 7200 acaactgcct cgaagccgcc aggttgtctc ttgagcaagc tcttgctggg atgggccaaa 7260 cttgtgatct cacagctgct gaggtggaaa agctaaagcg tatcatcagt caacttcaag 7320 gtttgaccac tgagcaagct ttaaactgtt agccgccagc ggcttgaccc gctgtggccg 7380 cggcggctta gttgtgactg aaacggcggt gaaaattata aaataccaca acagaacttt 7440 caccttgggc cctttagacc ttaaagtcac ttccgaggtg gaggtgaaga aatcaactga 7500 gcagggccac gctgttgtgg caaatttatg ttccggtgtc gttttgatga gacctcaccc 7560 accgtccctt attgacgttc ttctgaaacc cggacttgac acgacacccg gcattcaacc 7620 ggggcatggg gccgggaata tgggcgtgaa cggctctact tgggactttg aaactgcgcc 7680 cacaaaggca gaactcgagt tatccaagca aataattcaa gcatgtgaag ttaggcgcgg 7740 ggatgccccg aacctccaac tcccttacaa gctctatcct gttagggggg atcctgaacg 7800 gcataggggc cgcctcatca acaccaggtt tggagatttg tcctacaaaa ctcctcaaga 7860 caccaagtcc gcaatacatg cggcttgttg cctacatccc aacggggccc ccgtgtctga 7920 tggtaaatcc acattaggca ccactcttca acacggtttt gagctttatg tccctactgt 7980 gccctacagt gtcatggagt accttgattc acgccctgac acccctttca tgtgcactaa 8040 acatggcact tccaaggctg ctgcagagga tctccaaaaa tatgacctgt ccacccaagg 8100 gtttgtccta cctggggttc tgcgcctagt gcgcagattc atcttcggcc acatcggtaa 8160 agcgccgcca ttgttccttc catcaaccta tcccgccaag aactctatgg cagggatcaa 8220 tggccagaga ttcccaacaa aggacgtcca gagcatacct gaaattgatg agatgtgtgc 8280 ccgcgccgtc aaagagaatt ggcaaactgt gacaccttgc accctcaaga agcagtactg 8340 ttctaagcct aaaaccagga ccatcctggg caccaacaac ttcattgccc tggctcacag 8400 atcagcgctc agtggcgtca cccaggcatt catgaagaag gcttggaggt ccccaattgc 8460 cttggggaaa aataaattca aggaactgca ttgtactgtt gccggtaggt gccttgaggc 8520 cgacttagct tcctgtgacc gcagcacccc cgccattgta cgatggtttg ttgctaacct 8580 cctgtatgaa cttgcgggat gtgaagagta tctgcccagc tacgtgctta actgctgtca 8640 tgaccttgtg gcaacacagg atggtgcctt cacgaaacgt ggtggcctgt catccgggga 8700 ccccgttacc agtgtgtcta acaccgtgta ttcactggtg atctatgccc agcacatggt 8760 attgtcagcc ttgaaaatgg gtcatgagat tggtcttaag ttcctcgagg aacagcttag 8820 attcgaggac ctccttgaaa ttcaacccat gttggtatac tctgacgacc ttgtcttgta 8880 tgctgaaaaa cccacttttc ccaattacca ttggtgggtc gagcaccttg acttgatgct 8940 gggttttaaa acggacccga agaaaactgt cataactgac aagcccagct tccttggctg 9000 caggattgag ggagggcgac aactagtccc caatcgcgac cgcattctcg ccgctcttgc 9060 ataccacatg aaggcacaga atgcttcaga gtattatgca tctgctgccg caatcctgat 9120 ggattcatgt gcttgcattg accatgaccc tgagtggtat gaggatctta tttgcggcat 9180 tgccaggtgc gctcgccaag atggctacag ttttcccggc ccggcatttt ttatgtccat 9240 gtgggaaaag ctgaaaagtc acaatgaggg aaaaaaattc cgccactgcg gcatctgtga 9300 cgccaaagcc gaccatgcgt ccgcctgtgg acttgatttg tgtttgttcc actcgcattt 9360 tcaccagcac tgcccagtca ctctgggttg tggccaccat gccggttcaa aggaatgcca 9420 gcagtgccag tcacctgttg gagctggcag gtcccctctt gataccgtgc ttaaacaaat 9480 cccgtataaa cctcctcgca ctgttatcat gaaggtggat aataagacga cagccctcga 9540 tccaggaaga tatcagtccc gtcgaggtct cgttgcagtc aagaggggta ttgcaggcaa 9600 tgaagttgat cttgctgatg gagactacca ggtagtgccc cttttgccga cttgcaaaga 9660 cataaatatg gtgaaggtgg cttgcaatgt gctactcagc aagttcatag taggaccacc 9720 aggttccgga aagactacct ggttgttgag tcaggtccag gacgatgatg tcatctacac 9780 acccacccat cagaccatgt ttgatatagt cagtgctctc aaagtttgca ggtattctgt 9840 tccaggagcc tcaggactcc cttttccacc acccgccaga tccgggccgt gggttaggct 9900 cattgccagc gggcacattc ccggccgagt atcgtacctc gatgaggcag gatattgtaa 9960 tcatctggac atccttagac tgctttccaa aacacccctt gtgtgcttag gtgaccttca 10020 gcaacttcac cctgtcggct tcgattccta ctgttatgtt tttgatcaaa tgcctcagaa 10080 gcagctgacc actatttata gatttggccc caacatctgc gcagccatcc agccctgtta 10140 cagggaaaaa cttgaatcca aggctaggaa caccagggtg gtttttacca cccggccagt 10200 gacctttggt caggtactga caccatacca taaagatcgc gttggctctg caataaccat 10260 cgactcatcc caaggggcca cctttgatgt tgtgacatta cacctgccat ccccgaagtc 10320 cctgaataaa tcccgagcac ttgtagccat tactcgggcg agacacgggt tgttcattta 10380 cgacccccat ggccagctcc aggagttttt taacctgagc cctgagcaca ctgactgcaa 10440 ccttgtgttc agccgtgggg atgagctggt agttctgaat gcggacaatg cggtcacaac 10500 tgtagcgaag gccctagaga cgggtccgtc ccgcttccga gtgtcagacc cgagatgcaa 10560 gtctctttta gccgcttgct cggccagtct ggaggggagc tgcatgccac taccacaagt 10620 ggcacataac ctggggtttt acttttcccc cgacagccca gtatttgcac ctcttccaaa 10680 agagttggca ccgcattggc cagtggttac ccaccagaat aatcgggcgt ggcctgatcg 10740 gctcgttgct agcatgcgcc caattgatgc ccgctacagc aaaccaatgg tcggtgcagg 10800 gtatgtagtc gggccgtcca cctttctcgg cacccccggt gtggtgtcat actatctcac 10860 actgtacgtc aagggcgagc cccaggcctt gccggaaaca cttgtttcaa cagggcgtat 10920 agccacggac tgtcgggagt atctcgacac ggctgaggag gaggcagcaa aagaactccc 10980 ccacgcattt attggcgatg tcaaaggtac caaggtcggg ggatgtcatc acatcacatc 11040 aaaatacctg cctaggtttc tgcccaagga ctccgttgcc gtagttggag taagttcgcc 11100 cggcaaagct gctaaagccg tgtgcactct caccgatgtg tacctccctg aacttcggcc 11160 gtatttgcaa cctgagacgg catcgaaatg ctggaaactc aagttagact tcagggacgt 11220 tcgattgatg gtctggaaag gagccactgc ctacttccaa ttggaagggc tcacatggtc 11280 ggcgttgccc gattatgcca ggtttattca gctgcccaaa gacgccattg tatacattga 11340 tccatgtata ggaccggcga cggccaaccg taaggtcgtg cgaaccacgg actggcgggc 11400 cgacctggca gtgacaccgt atgattacgg ggcccagtac attttgacaa cagcctggtt 11460 cgaggacctc gggcctcagt ggaaaatttt ggggttgcag ccctttaggc gggcatttgg 11520 ctttgaaaac accgaggact gggcaatcct tgcgcgccgt atgaatgacg gcaaggacta 11580 cattgactac aactggaatt gcgtccgagg acgcccacac gctatccacg ggcgcgctcg 11640 tgaccatacc tatcattttg ccttgggcac ggaattgcag gtggagctgg gtaaacccga 11700 ctgccgcctg agctagcacc gtgaacctga agtgatgcaa tggggtcgtt gtggagtaaa 11760 attagccagt tgtttgtgga cgccttcacg gagtttcttg ttagtgtggt tgatatcgtc 11820 atctttcttg cgatactgtt tgggttcacc gtcgcaggat ggttattggt ctttcttctc 11880 agattggttt gctccgcgat tctccgttcg cgctctgcca ttcactctcc cgaactatcg 11940 aaggtcttat gaaggcttgc tacccaattg cagaccggac gtcccacaat tcgcattcaa 12000 acacccgttg ggtatgcttt ggcacatgcg ggtctcccac ttaattgatg agatggtctc 12060 tcgccgcatt taccagacta tggaacattc aggtcaagcg gcctggaagc aggtagttgg 12120 tgaggccact ctcacgaagc tatcaaagct cgacatagtc acccatttcc aacacctggc 12180 cgcagtggag gcggattctt gccgctttct tagctcacga ctcgtgatgc taaagaatct 12240 tgtcgttggc aatgtaagcc tacaatacaa caccacgttg gaccacgttg agctcatttt 12300 ccccactcca ggcacgaggc ccaagttgac cgatttcaga caatggctga tcagtgtaca 12360 tgcttccatt ttttcctctg tagcttcatc tgtcactttg ttcgtggtgc tttggcttcg 12420 agttccaatt ctacgctatg tttttggttt ccattggctc acggcaacac atcattcgag 12480 ctaaccatca attacaccat atgcatgccc tgccgcacca gtcaagcggc taaacaaagg 12540 cttgaacccg gtcatagcat gtggtgcaaa atagggcaca ccagctgtga ggagagtgac 12600 cacgatgagt tgttaatgac tatcccgtcc gggtacgata acctcaaact tgagggctat 12660 tatgcttggc tagccttctt gtccttttcc tacgcggccc aatttcatcc ggagctgttt 12720 ggaataggga atgtgtcgcg cgtcttcgtg gacaagcgac accaggccat ttgtgcggtg 12780 catgatggac tcaattcaac cgtacccatt aatcacaaca tctccgcatc gtatgcggtg 12840 tactatcatc atcaaataga cggaggcaat tggttccacc tagaatggct gcggccattc 12900 ttttcctcct ggttggtgct caacgtctca tggtttctga ggcgttcgcc tgcaagccct 12960 gcttctcgac gcatttatca gatattgaga ccaacacgac cgcggctgcc ggtttcatgg 13020 tccttcagga aatcgaatgt ctccagcctc acagggactc agcagcgcag cagagcgttc 13080 cctataagaa acagtcgcaa tgtcgccaag ccgtcggcac cccccagtac atcacgataa 13140 cggctaatgt gaccgacgaa tcatacttgt ataacgcgga tctgctgatg ctttccgcgt 13200 gccttttcta cgcctcagaa atgagcgaga aaggcttcaa agttatcttt gggaatgtct 13260 ctggcgttgt ttccgcttgc gtcaatttca cagattacgt ggcccatgtg acccaacaca 13320 cccagcagca ccatctggta attgatcatg tccgattact gcatttcctg tctccatcta 13380 caatgaggtg ggccacaacc atcgcttgtt tgttctccat tctcttggcg atatgagatg 13440 ttctcacaaa ttggggtgtt tcttgattcc gcactcttgc ttttggtggc tttttttgct 13500 gtgtatcggc ttgtcctggt cctttgccga tggcaacggc aacagctcga cataccaata 13560 catatataac ttgacgatat gcgagctgaa tgggaccgcc tggttgtcta cccacttttc 13620 ttgggcagtc gagacctttg tgctttaccc ggttgcgact cacattctct cactgggttt 13680 tctcacaaca agccatttct ttgatgcgct cggtctcggc gctgtgtcca tcacagggtt 13740 ttgtggcaaa cgatatgtgc tcagcagtat ctacggcgct tgtgctcttg cagcgttcgt 13800 gtgcttcgcc atccgtgctg ctaaaaattg tatggcttgc cgctacgccc gcacccggtt 13860 caccaacttc attgtagacg accgggggag gattcatcgg tggaagtccc cggtggtggt 13920 ggagaaattt ggcaaagccg aaattggcgg cggtcttgtc accatcaaac atgtcgtcct 13980 cgaaggggtt aaagctcaac ccttgacgag gacttcggct gagcaatggg aagcctagac 14040 agtttttgcg atgatcctgc tgctgtgcaa aagcttgtgc tagcctttag cattacatac 14100 acacctataa tgatatatgc ccttaaggtg tcacgcggtc ggctcttagg gctgctgcac 14160 atcctgatat tcctgaactg ctcctttact ttcgggtaca tgacgtatgt gcacttccaa 14220 tccgccaacc gtgtcgcact cactttgggg gccgttgttg ccctcctgtg gggcgtttat 14280 agcttcacag agtcatggaa gttcattact ttcagatgca gattgtgttg cctaggccgg 14340 cgatacattc tggcccctgc ccaccacgta gaaagtgctg caggtctcca ttcaatccca 14400 gcgtctggta accgagcata cgctgtgaaa aagcccggac taacatcagt aaacggcact 14460 ctagtaccag gacttcggag cctcgtgctg ggcggcaaac gagctgttaa acgaggagtg 14520 gttaacctcg tcaagtatgg ccggtagaaa ccagagccag aagaaaaaga gaaacacagc 14580 tccaatgggg aatggccagt cagtcaatca actgtgccaa ttgctgggta caatgataaa 14640 gtcccagcac caacggtcca ggggaggaca ggctaaaaag aaaaagcctg agaagccaca 14700 ttttcccttg gctgctgaag atgacatccg gcatcacctc acccaaactg aacgttccct 14760 ctgcttgcaa tcgatccaga cggctttcaa tcaaggcgca ggaactgcgt cgctttcatc 14820 cagtgggaag gtcagttttc aggttgagtt catgttgccg gttgctcata cagtgcgctt 14880 gattcgcgtg acttctacat ccgccagtca ggatgcaaat taacttgaca gtcaggtgaa 14940 tggccgcgat tggcgtgtgg cctctgagtc acctattcaa ttagggcgat cacatggggg 15000 tcatacttaa ttaggcaaga accatgtgac cgaaattaaa aaaa 15044 <210> 2 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> E38_ORF7_original <400> 2 atggccggta gaaaccagag ccagaagaaa aagagaaaca cagctccaat ggggaatggc 60 cagtcagtca atcaactgtg ccaattgctg ggtacaatga taaagtccca gcaccaacgg 120 tccaggggag gacaggctaa aaagaaaaag cctgagaagc cacatttcc cttggctgct 180 gaagatgaca tccggcatca cctcacccaa actgaacgtt ccctctgctt gcaatcgatc 240 cagacggctt tcaatcaagg cgcaggaact gcgtcgcttt catccagtgg gaaggtcagt 300 tttcaggttg agttcatgtt gccggttgct catacagtgc gcttgattcg cgtgacttct 360 acatccgcca gtcaggatgc aaattaa 387 <210> 3 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> E38_ORF7_CPD7_O <400> 3 atggccggta gaaaccagag ccagaagaaa aagagaaaca cagctccaat ggggaatggc 60 cagtcagtca atcaactgtg ccaattgctg ggtacaatga taaagtccca gcaccaacgg 120 tccaggggag gacaggctaa aaagaaaaag cctgagaagc cacatttcc cttggccgcc 180 gaagatgaca tccggcatca ccttacccaa acggaacgtt cgctctgctt gcaatcgatc 240 cagacggctt tcaatcaagg cgccggtact gcgtcgcttt catccagtgg gaaggtcagt 300 tttcaggttg agttcatgtt gccggttgct catacagtgc gcttgattcg cgtgacttct 360 acatccgcca gtcaggatgc aaattaa 387 <210> 4 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> E38_ORF7_CPD22_X <400> 4 atggccggta gaaaccagag ccagaagaaa aagagaaaca cagctccgat ggggaatggc 60 cagtcagtca atcaactgtg ccaattgctg ggtacaatga taaagtcgca acaccaacgg 120 tctaggggcg gacaggctaa aaagaaaaag cccgagaagc cgcattttcc gttagccgcc 180 gaagacgata tccggcatca ccttacccaa acggaacgtt cgctctgctt gcaatcgatc 240 cagacggctt tcaatcaagg cgccggtact gcgtcgcttt catctagcgg taaggttagt 300 tttcaggttg agttcatgtt gccggttgct catacagtgc gcttgattcg cgtgacttct 360 acatccgcca gtcaggatgc aaattaa 387 <210> 5 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> E38_ORF7_CPD14_NUM_O <400> 5 atggccggta gaaaccagag ccagaagaaa aagagaaaca cagctccaat ggggaatggc 60 cagtcagtca atcaactgtg ccaattgctg ggtacaatga taaagtccca gcaccaacgg 120 tctaggggcg gacaggctaa aaagaaaaag cctgagaagc cacattttcc cttggccgcc 180 gaagacgata tccggcatca ccttacccaa acggaacgtt cgctctgctt gcaatcgatc 240 cagacggctt tcaatcaagg cgccggtact gcgtcgcttt catccagcgg taaggttagt 300 tttcaggttg agttcatgtt gccggttgct catacagtgc gcttgattcg cgtgacttct 360 acatccgcca gtcaggatgc aaattaa 387 <210> 6 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> E38_ORF7_CPD16_NUM_X <400> 6 atggccggta gaaaccagag ccagaagaaa aagagaaaca cagctccaat ggggaatggc 60 cagtcagtca atcaactgtg ccaattgctg ggtacaatga taaagtcgca acaccaacgg 120 tctaggggcg gacaggctaa aaagaaaaag cctgagaagc cacattttcc cttggccgcc 180 gaagacgata tccggcatca ccttacccaa acggaacgtt cgctctgctt gcaatcgatc 240 cagacggctt tcaatcaagg cgccggtact gcgtcgcttt catccagcgg taaggttagt 300 tttcaggttg agttcatgtt gccggttgct catacagtgc gcttgattcg cgtgacttct 360 acatccgcca gtcaggatgc aaattaa 387 <210> 7 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> E38_ORF7_CPD18_NUM_X <400> 7 atggccggta gaaaccagag ccagaagaaa aagagaaaca cagctccaat ggggaatggc 60 cagtcagtca atcaactgtg ccaattgctg ggtacaatga taaagtccca gcaccaacgg 120 tctaggggcg gacaggctaa aaagaaaaag cccgagaagc cgcattttcc gttagccgcc 180 gaagacgata tccggcatca ccttacccaa acggaacgtt cgctctgctt gcaatcgatc 240 cagacggctt tcaatcaagg cgccggtact gcgtcgcttt catccagcgg taaggttagt 300 tttcaggttg agttcatgtt gccggttgct catacagtgc gcttgattcg cgtgacttct 360 acatccgcca gtcaggatgc aaattaa 387 <210> 8 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> E38_ORF7_CPD20_NUM_X <400> 8 atggccggta gaaaccagag ccagaagaaa aagagaaaca cagctccaat ggggaatggc 60 cagtcagtca atcaactgtg ccaattgctg ggtacaatga taaagtcgca acaccaacgg 120 tctaggggcg gacaggctaa aaagaaaaag cccgagaagc cgcattttcc gttagccgcc 180 gaagacgata tccggcatca ccttacccaa acggaacgtt cgctctgctt gcaatcgatc 240 cagacggctt tcaatcaagg cgccggtact gcgtcgcttt catccagcgg taaggttagt 300 tttcaggttg agttcatgtt gccggttgct catacagtgc gcttgattcg cgtgacttct 360 acatccgcca gtcaggatgc aaattaa 387 <210> 9 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> E38_ORF7_CPD18_POSITION_O <400> 9 atggccggta gaaaccagag ccagaagaaa aagagaaaca cagctccaat ggggaatggc 60 cagtcagtca atcaactgtg ccaattgctg ggtacaatga taaagtccca gcaccaacgg 120 tctaggggcg gacaggctaa aaagaaaaag cctgagaagc cacattttcc cttggccgcc 180 gaagacgata tccggcatca ccttacccaa acggaacgtt cgctctgctt gcaatcgatc 240 cagacggctt tcaatcaagg cgccggtact gcgtcgcttt catccagcgg taaggttagt 300 tttcaggtcg agtttatgtt gccggtcgcg catacagtgc gcttgattcg cgtgacttct 360 acatccgcca gtcaggatgc aaattaa 387 <210> 10 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> E38_ORF7_CPD22_POSITION_O <400> 10 atggccggta gaaaccagag ccagaagaaa aagagaaaca cagctccaat ggggaatggc 60 cagtcagtca atcaactgtg ccaattgctg ggtacaatga taaagtccca gcaccaacgg 120 tctaggggcg gacaggctaa aaagaaaaag cctgagaagc cacattttcc cttggccgcc 180 gaagacgata tccggcatca ccttacccaa acggaacgtt cgctctgctt gcaatcgatc 240 cagacggctt tcaatcaagg cgccggtact gcgtcgcttt catccagcgg taaggttagt 300 tttcaggtcg agtttatgtt gccggtcgcg catacggtac gcttgatacg cgttacttct 360 acatccgcca gtcaggatgc aaattaa 387 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PRRSV_forward <400> 11 tggccagtca gtcaatcaac 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PRRSV_reverse <400> 12 aatcgattgc aagcagaggg aa 22

Claims (8)

In the ORF7 region consisting of the nucleic acid sequence of SEQ ID NO: 2 in the genome of wild-type porcine reproductive and respiratory syndrome virus
The 123rd C is substituted with T, A at the 129th position is substituted with C, the 177th T is substituted with C, the 180th T is substituted with C, the 186th T is substituted with C, the 189th C is substituted with T, The 204th C is substituted with T, the 213th T is substituted with G, the 222th C is substituted with G, the 264th A is substituted with C, the 267th A is substituted with T, the 288th T is substituted with C, the 291th G is substituted with T, 297th C is substituted with T, 309th T is substituted with C, 315th C is substituted with T, 327th T is substituted with C, 330th T is substituted with G, 336th A is introducing a nucleotide sequence introducing a silent mutation comprising a mutation in which G is substituted, G at 339 is substituted with A, T at position 348 is substituted with A, and G at position 354 is substituted with T; and
A method for producing an attenuated porcine reproductive and respiratory syndrome virus, comprising the step of generating a virus into which the mutation is introduced. delete The method of claim 1 , wherein the porcine reproductive and respiratory virus is a European porcine reproductive and respiratory syndrome virus. A pharmaceutical composition for preventing porcine reproductive and respiratory syndrome (PRRS) comprising an attenuated porcine reproductive and respiratory syndrome virus mutant prepared by the method of any one of claims 1 and 3. 5. The composition of claim 4, wherein the porcine reproductive and respiratory syndrome is European porcine reproductive and respiratory syndrome. 5. The composition of claim 4, wherein the composition is a vaccine composition. 7. The composition of claim 6, wherein the vaccine is a live vaccine. A kit for preventing porcine reproductive and respiratory syndrome (PRRS) comprising an attenuated porcine reproductive and respiratory virus mutant prepared by the method of any one of claims 1 and 3.

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