KR20150045873A - Chimeric strain of Porcine Reproductive and Respiratory Syndrome Virus - Google Patents

Abstract

The present invention relates to a chimeric mutant of porcine reproductive and respiratory syndrome virus which can be used as a vaccine. The chimeric virus mutant of the present invention has low pathogenicity and high safety in pigs. It has a wide defensive range by expressing the nonstructural protein of the strongly toxic mutant and the dominant variant of the mutant strain in Korea and has a merit of obtaining a high antibody value even when used alone as the Sadox vaccine alone. Thus, it can be used to effectively defend the swine genital respiratory syndrome (PRRS), a swine disease that causes serious economic harm.

Description

{Chimeric strain of Porcine Reproductive and Respiratory Syndrome Virus in Porcine Reproductive Respiratory Syndrome Virus}

The present invention relates to a chimeric mutant of porcine reproductive and respiratory syndrome virus which can be used as a vaccine.

Porcine Reproductive and Respiratory Syndrome (PRRS) is the most common infectious disease in the domestic swine industry, along with swine fever virus infection and foot-and-mouth disease. PRRS causes symptoms of reproductive failure in abortion, premature birth, and stillbirth in sows, and respiratory symptoms such as sneezing and fever in piglets and finishing pigs. Generally, after infection with a virus, secondary infection such as bacteria causes severe respiratory symptoms. However, in case of chronic infection, reduction in body weight and increase in mortality are observed without characteristic clinical symptoms.

The causative agents of PRRS are Arterivirus , Arteriviridae, and PRRS virus belonging to the family Nidovirales. The PRRS virus has a positive-sense single-stranded RNA genome, approximately 15.4 kilobases in size. The genome of the PRRS virus has nine ORFs (Conzelmann et al., 1993; Meulenberg et al., 1993). Of these, ORF1a and ORF1b, which encode Non Structural Protein (NSP), account for about 80% of the viral genome (Bautista et al., 2002; Meulenberg et al., 1993; Snijder and Meulenberg, 1998, 2001). The glycosylated structural proteins GP2, GP3, GP4, and GP5, the non-glycosylated membrane (Membrane, M) protein, and the nucleocapsid (N) protein are encoded by the ORF occupying the remaining 20%. The minor structural proteins GP2, GP3, and GP4 form a heterotrimer that acts when the virus enters the host cell. The major structural proteins, GP5 and M, act as a heterodimer to increase the infectivity of the virus.

The PRRS virus is highly mutated due to the nature of the RNA virus, and there is a large difference between the viruses. PRRS viruses are divided into North American and European types. Up to 40% of genetic differences exist between North American and European varieties. In addition, there is often no cross protection between mutants belonging to the same type (Meng, X. J. et al., 2000). Therefore, the standard mutation-based vaccine has been produced for each of them, but it has not been able to effectively prevent PRRS because of its poor cross-resistance. In order to overcome this, various attempts have been made to produce a vaccine having safety, immunogenicity, and protective ability effectively.

The present invention provides a chimeric mutant of porcine reproductive and respiratory syndrome virus which can be used as a vaccine, a vaccine containing the vaccine, and a method for producing the same.

In order to achieve the above object, the present invention provides DNA of a porcine reproductive and respiratory syndrome virus (PRRSV) chimeric mutant having the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the mutant strain having the nucleotide sequence shown in SEQ ID NO: 1 was named K418. In SEQ ID NO: 1, 1-12160 and 15467-19231 are PRRSV FL12 (infectious clones in which the mutant PRRSV NVSL 97-9785 is inserted into the pBR322 vector) and 12161-15466 are in the PRRSV LMY sequence.

The present invention provides an RNA reverse transcribed from a DNA having the nucleotide sequence shown in SEQ ID NO: 1.

The present invention also provides DNA of a porcine reproductive and respiratory syndrome virus (PRRSV) chimeric mutant having the nucleotide sequence shown in SEQ ID NO: 2.

In the present invention, the mutant strain having the nucleotide sequence shown in SEQ ID NO: 2 was named K418DM. The other part in SEQ ID NO: 2 is the same as SEQ ID NO: 1, and there is a difference in that the region corresponding to the regions coding for GP5 protein, 13886-13888 and 13940-13942, was mutated and deglycosylated.

The present invention provides RNA reverse transcribed from the nucleotide sequence DNA of SEQ ID NO: 2.

The present invention provides a cell comprising the DNA of a porcine reproductive and respiratory syndrome virus (PRRSV) chimeric mutant having the nucleotide sequence shown in SEQ ID NO: 1.

The present invention provides a cell comprising the DNA of a porcine reproductive and respiratory syndrome virus (PRRSV) chimeric mutant having the nucleotide sequence shown in SEQ ID NO: 2. The cell may be a cell with accession number KCCM 11458P. The cells may also be Escherichia coli DH5a cells.

The present invention provides a porcine reproductive and respiratory syndrome virus vaccine comprising the DNA of the porcine reproductive tract respiratory syndrome virus chimeric mutant described in SEQ ID NO: 1 or SEQ ID NO: 2 or RNA reverse transcribed therefrom.

The vaccine may further comprise at least one member selected from the group consisting of carriers, diluents, excipients, and adjuvants.

The virus may be inactivated.

The present invention provides a method for producing porcine reproductive / respiratory syndrome virus chimeric mutants using the cells.

The present invention provides a method for producing a porcine reproductive-respiratory syndrome virus vaccine using the cell.

The present invention provides a method for producing a porcine reproductive-respiratory syndrome virus vaccine using the gene.

K418 of the present invention includes ORF1 in NVSL 97-9785 and ORF7 in ORF2 of LMY.

The present invention provides a method for producing a porcine reproductive / respiratory syndrome virus chimeric mutant having the nucleotide sequence of SEQ ID NO: 1 by combining the ORF1 region of the PRRSV NVSL 97-9785 mutant and the ORF2 to ORF7 of the PRRSV LMY mutant.

Also,

1) combining the ORF1 region of the PRRSV NVSL 97-9785 mutant and the ORF2 to ORF7 of the PRRSV LMY mutant; And

2) producing a porcine reproductive / respiratory syndrome virus chimeric mutant having the nucleotide sequence of SEQ ID NO: 2, comprising the step of causing a mutation in the gene encoding GP5 in the combined base sequence.

The chimeric virus mutant of the present invention has low pathogenicity and high safety in pigs. It has a wide defensive range by expressing the nonstructural protein of the strongly toxic mutant and the dominant variant of the mutant strain in Korea and has a merit of obtaining a high antibody value even when used alone as the Sadox vaccine alone. Thus, it can be used to effectively defend the swine genital respiratory syndrome (PRRS), a swine disease that causes serious economic harm.

Figure 1 shows the phylogenetic tree of PRRS virus by GP5 protein.
Figure 2 shows a general genomic arrangement of PRRS viruses.
FIG. 3 is a schematic diagram showing a genomic arrangement of K418 synthesized by cutting and connecting fragments of FL12 clones and LMY mutants, respectively.
FIG. 4 shows the results of immunofluorescence analysis using an antibody specific to a nucleocapsid protein (N protein) of PRRSV in MARC-145 cells transfected with K418.
5 is a graph showing the analysis of the multi-step growth kinetics of FL12, LMY and K418.
FIG. 6 is a graph showing the generation of a neutralizing antibody by K418DM in an outdoor test.

Since the RNA is easily destroyed, it is converted into DNA and all the work is carried out. Then, RNA is synthesized from this and the cells are transfected.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

The reagents and solvents referred to hereafter will be purchased from the Sigma Aldrich ^® unless otherwise specified.

Example  One. Chimeric  Production of viruses

1-1. Chimeric  Design of viruses

The PRRS chimeric virus was designed to include the ORF1 region of the North American isolate NVSL 97-9785 and the ORF2 to ORF7 region of the most prevalent mutant LMY in Korea. NVSL 97-9785 is known to be an insect causing sodic acid in sows (Allende et al., 2000), and LMY is a representative mutant in Korea and is not known to be pathogenic (Cha SH et al., 2006).

The GP5 protein of the PRRS virus has the highest mutation rate among the isolates and plays an important role because it contains a neutralizing antibody epitope that is involved in the neutralization ability of the virus and is also used to classify the PRRS virus system. The phylogenetic tree of the GP5 protein of the PRRS virus used in isolates and vaccines of each country is shown in Fig. The bottom bar means that there are 0.05 nucleotide substitutions per nucleotide position. In FIG. 1, NVSL 97-9785 and LMY are mutants having a genetic relationship of about 85% with each other.

1-2. NVSL  97-9785 Variant and LMY  Preparation of mutant strains

Dr. of Lincoln University, Nebraska, USA Fernando Osorio and Dr. From Asit Pattnaik, pFL12, a cDNA clone of FL12, an infectious clone in which all the genes of the NVSL 97-9785 mutant were inserted, was used.

The LMY mutant, the most prevalent mutant in Korea, was purchased from the Ministry of Agriculture, Forestry and Fisheries Inspection, and the genome region containing the entire structural gene of LMY was amplified by reverse transcription PCR using the primers shown in Table 1 below.

1-3. Chimeric  Production of viral clones

As shown in the schematic diagram of Fig. 2, the PRRS virus contains a total of 8 ORFs, i.e. ORF1a, ORF1b, and ORF2-7.

In the genomic region containing the entire structural gene of the LMY mutant amplified in 1-2, the region from ORF2 to ORF7 was digested with restriction enzymes ECoRV and PacI, and the plasmid was prepared by substituting the corresponding region of pFL12 K418. A schematic diagram of K418 is shown in Fig.

In the K418 plasmid, two of the three glycosylation sites (34 and 51 amino acids) present in the eptodomain of the GP5 moiety in the GP5-encoding genomic region were subjected to site-directed mutagenesis to produce another amino acid The N-glycosylation site of GP5 was removed by substitution. GP5 plays an important role in the virus neutralizing ability, and it can induce active antibody production by exposing an epitope involved in neutralizing antibody formation through deglycosylation of GP5. The variant plasmid thus constructed was named K418DM. All-length variant plasmids were extracted and prepared using the QIAprep Spin Midiprep kit (Qiagen, Korea).

1-4. Chimeric  virus RNA Warrior  synthesis

The K418 plasmid and K418DM plasmid prepared in 1-3 were linearized by treatment with AclI. Each RNA transcript was synthesized in vitro using mMESSAGEmMACHINE Ultra T7 kit (Ambion, Austin, Tex.) Using the prepared linear DNA as template.

1-5. Chimeric  virus RNA Transfection of transfection )

MARC-145 cells, known as PRRS virus-soluble cell lines, were used to confirm the replication of chimeric viruses. MARC-145 cells were purchased from Nebraska-Lincoln University.

MARC-145 cells were suspended in PBS buffer. MARC-145 cells were electroporated at 250V and 975 uF using Gene Pulser Xcell (Bio-Rad, Hercules, Calif.). RNA was isolated from the naive MARC-145 cells not infected with PRRSV to the cells themselves. This RNA acts as a carrier to help infect viral RNA. About 5 ug of RNA against the naive MARC-145 cell itself and about 10 ug of RNA transcripts of K418 and K418DM were transfected into the perforated MARC-145 cells with a 4.0 mM cuvette, respectively.

Transfected MARC-145 cells were diluted in DMEM medium containing 10% FBS and 1.25% DMSO and dispensed into 6 well plates.

After 48 hours of transfection, it was confirmed that K418 and K418DM viruses were amplified in MARC-145 cells by immunofluorescence assay (IFA) using SDOW17 antibody targeting PRSPV nucleoprotein (FIG. 4) .

1-6. chimera  Virus multi - step growth kinetics

MARC-145 cells were cultured in a 96-well plate two days before measurement of potency, and FL12 and LMY, parental viruses, and K418, a chimeric virus of the present invention, were diluted with 10-fold dilutions of the parental viruses into MARC-145 cells, After 12, 24, 48, 72, and 96 hours of infection, culture supernatants were collected and multi-step growth kinetics were compared by measuring virus titers using the Reed and Muench method (FIG. 5). As can be seen from FIG. 5, K418 showed a growth pattern similar to that of FL12, LMY, and it was found that genetic manipulation applied to K418 did not significantly affect virus growth. K418 showed similar final titers to FL12, LMY, but virus growth was slightly delayed.

1-7. Chimeric  Virus-induced Sadoc vaccine  making

1-5 cells were transfected with MARC-145 cells for 10 days to confirm PRRS virus-specific cytopathic effect (CPE). Transfected supernatant produced 80% CPE on day 5 of infection and the calculated virus turnover was 10 ⁶ TFID ₅₀ / ml.

The cell culture flask infected with the virus was frozen in a -70 degree freezer and dissolved at room temperature. The cells were centrifuged three times to recover the supernatant from which cells were removed, and K418 and K418DM were recovered therefrom.

The virus was inactivated according to the following procedure to make a vaccine.

The virus was inactivated by adding 1 mM BEI (binary ethylenimine) to K418 and K418DM, respectively, and incubating at 37 DEG C for 24 hours. After that, 0.1 mM Na-thiosulphate was added to neutralize BEI and left at 37 ° C for 2 hours.

The following procedure was performed to confirm whether the virus was inactivated. The MARC-145 cells were proliferated in a tissue culture flask for 2 days, then the cell culture medium was removed, and the inactivated K418 and K418DM were inoculated and sensitized at 37 ° C for 1 hour. After that, fresh medium was added and cultured. Three days after the incubation, the amplification of the virus was confirmed by immunofluorescence analysis using SDOW17 antibody targeting PRSPV nucleoprotein, and it was observed whether a PRRS virus-specific cytopathic effect (CPE) appeared after 7 days .

Immunofluorescence analysis showed that the virus was not amplified and CPE was not observed, so that K418 and K418DM were successfully inactivated. In particular, CPE observations did not reveal PRRS virus specific CPE as well as CPE caused by other viruses and cytopathic effects due to other reasons.

Example  2. MLV  And NVSL97 With -9785 Homology  compare

To investigate the relationship between the K418 synthesized in Example 1 and the parental virus of the in-gel PRRS virus vaccine (MLV), K418 of the domestic virulent vaccine host Boehringer Ingelheim, and NVSL97-9785, .

The genomic region corresponding to GP5 protein of K418 was amplified by RT-PCR. Primers used for ORF5 of PRRSV were used as primers. Table 1 below shows the GP5 protein gene base sequence of K418 of the present invention.

[Table 1]

As a result of comparing GP5 protein gene sequences of K418, MLV, and NVSL97-9785, MLV and K418 showed about 94% homology with each other. In addition, NVSL97-9785 and K418 showed about 89% homology with each other. The results of comparative analysis of homology are shown in Table 2 below.

[Table 2]

Example  3. Cell Substrate Study

When K418 and K418DM prepared in Example 1 were transferred, it was confirmed how long the modified region was studied.

As a result, the deformed region was maintained up to about 9 times in K418DM, but the deformed region was not maintained consistently thereafter. This is because the virus tends to return to its original state and is highly mutated due to the nature of the RNA virus. Therefore, it is necessary to confirm whether or not the deformed portion is maintained each time the transmission occurs.

 The virus used in the vaccine was inoculated on MARC-145 cells, cultured at 37 ° C for 5 days, and then transferred to 2 times. The genes were identified by sequencing and stored at -70 ° C.

Example  4. In the pig  Pathogenicity confirmation

The pathogenicity of the K418 and K418DM viruses was investigated in 4 week old PRRS virus-negative pigs (purchased from PRRSV infected farms in Pocheon, Gyeonggi-do).

Twelve 4 - week - old pigs were immunized against the PRRS virus. Eight of them were inoculated with the vaccination group and four of them were the negative control group. Dividing the attack inoculation group back to 4 rats of two groups, one group is the K418, were intramuscularly K418DM the one group, the increments 10 ^4.5 TCID ₅₀ / ml, respectively.

Clinical symptoms were observed for one week after the attack.

In addition, blood was sampled after the inoculation and the serum was separated, and then MARC-145 cells were inoculated. Two days later, immunofluorescence analysis was performed to measure viremia in the serum.

As a result, all of the inoculated group showed clinical symptoms accompanied by fever and respiratory symptoms, but no clinical symptoms were observed in the negative control group. The virus in the sera of the inoculated group was also measured at 10 ^{3 to 4} TCID ₅₀ / ml. In the inoculated group, the K418 inoculated group had higher clinical symptoms and viremia in serum than the K418DM inoculated group (see Table 3).

[Table 3]

Example  5. Safety test

5-1. Sadoc vaccine  making

In Example 4, K418DM virus with lower pathogenicity was selected as a vaccine strain and its safety as a vaccine strain was evaluated. The inoculated vaccine was prepared by mixing the inactivated K418DM virus 10 ⁷ TCID ₅₀ / ml with Montanide IMS1313 at a ratio of 70:30 (w / w). Using this, safety tests in mice, guinea pigs and pigs were carried out.

5-2. Safety testing in mice

0.5 ml of the Sadox vaccine prepared in 5-1 was inoculated into the abdominal cavity of eight mice having a weight of about 20 g and observed for 7 days. The test results are shown in Table 4 below. All of them survived without symptoms for more than 7 days after vaccination.

[Table 4]

5-3. In Guinea  Safety test

2 ml of guinea pigs weighing about 300 g were inoculated intramuscularly with 2 ml of the Sadox vaccine prepared in 5-1, and the same amount was inoculated into the other 2 mice for 7 days. The test results are shown in Table 5 below. All of them survived without symptoms for more than 7 days after vaccination.

[Table 5]

5-4. In the pig  Safety test

Six healthy four - week - old pigs with negative PRRS virus antibody were inoculated with 5 - 1 Sadox vaccine. It was confirmed that there was no hypersensitivity reaction within 1 ~ 2 hours. During the test period, necrosis of the vaccine injection site, fever symptoms and respiratory symptoms were not observed. The test results are shown in Table 6 below.

[Table 6]

Example  6. In the pig  Confirm immunogenicity

In order to test the minimum immunogenicity of K418DM, various amounts of the antigen were inoculated and the activity of the produced antibody was measured. The inactivated K418DM virus was mixed with Montanide IMS1313 at a ratio of 70:30 (w / w) or 50:50 (w / w) to prepare a Sadox vaccine.

Various concentrations of K418DM were administered to the pigs and blood was collected after 4 weeks to separate the serum. Separated sera were denatured at 56 ° C for 30 minutes and then diluted with the medium. The same amount of PRRS virus (200 TCID ₅₀ /0.1 ml) was mixed and reacted at 37 ° C for 1 hour. This was inoculated into MARC-145 cells and cultured at 37 ° C. After 3 days of incubation, the mixture was immobilized on a 1: 1 mixture of methanol: acetone and ELISA kit (IDEXX), and antibody titers were measured by immunofluorescent staining with antibodies targeting N protein.

As a result, it was confirmed that the minimum immunogenicity was formed at an antigen amount of 10 ⁷ TCID ₅₀ / ml or more. Antibodies in the serum began to form from 2 weeks after the vaccination, and showed an increasing pattern over time. The highest antibody titers were found at 6 weeks of vaccination when the antigen dose of 10 ⁸ TCID ₅₀ / ml or more was administered. In the case of 10 ⁸ TCID ₅₀ / ml, the neutralization antibody level was more than 8 times higher than that of the Ministry of Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters (neutralized antibody 1: 4 or more).

The results of the minimum immunogenicity test of K418DM are shown in Table 7 below.

[Table 7]

Example  7. Outdoor Test

To evaluate the clinical efficacy of K418DM, a total of three pig farms were selected and field trials were conducted.

The domestic pig farm was divided into three major groups based on PRRSV: PRRSV uninfected farms, PRRSV open field, stabilized farms that did not develop lesions, and PRRSV infection history or PRRSV It is categorized as a farm using the in-gelback PRRS virulence vaccine (MLV) of the raw monologue Boehringer Ingelheim. In order to conduct an outdoor test, one farm was selected for each category (hereinafter referred to as A, B, and C farms, respectively). Twenty - two 4 - week - old piglets were selected for each of the farms, 18 of them were inoculated with K418DM, treated with K418DM inoculation group, and 4 were treated without negative treatment. After that, neutralization antibody production was analyzed for 7 weeks.

MARC-145 cells were mixed with PRRS K418DM and cultured for 2 days. Thereafter, the presence of neutralizing antibodies in serum collected from piglets was examined by FA assay. The antibody used was a monoclonal antibody (SDOW-17) specific for the PRRSV N protein as the primary antibody and an anti-mouse IgG FITC conjugated antibody (Alexa488) as the secondary antibody. The results are shown in Table 8 and FIG. 6 (the meanings of a, A, b, B, c and C are the same as those in Table 8). In the A farm, the positive rate of neutralizing antibody was about 38%, about 94% in B farm and about 72% in C farm, and it was confirmed that the effect of K418DM was significantly different depending on farm conditions.

The relatively low antibody positivity rate on A farms without PRRSV infection seems to be due to individual differences, and it is expected that more accurate results will be obtained if the population in the inoculation group is increased and then proceeded.

B farms showed high neutralizing antibody titers in most K418DM inoculated groups. In order to investigate the reason for the increase in immunogenicity by K418DM even though the piglets of the B farm had an antibody against the outdoor PRRSV which is present in the B farm, the nucleotide sequence of the GP5 protein portion of the open field and K418DM Were amplified by PCR and sequenced. As a result, it was found that there is about 96% high homology between GP5 protein, which is a virus structural protein contributing to immunogenicity of outdoor PRRSV, and GP5 protein of K418DM. From these results, it was found that the high neutralizing antibody level in the K418DM inoculation group of B farm was due to the vaccine effect on homologous mutants.

In the C farm, about 72% of the highly neutralized antibody was found in the K418DM vaccinated group. C farms were anticipated to have antibodies against most of the C pigs' piglets, as there were past PRRSV or attenuated live monospecific newborns that were either infected or currently infected. In order to clarify the reason for the further increase of immunogenicity by K418DM, the homology between GP5 protein and K418DM of PRRSV live monocytogenes inoculated at C farm was compared and it was confirmed that there was about 91% homology. From these results, it was found that the high neutralizing antibody level in the K418DM inoculation group of C farm was due to the vaccine effect on the homologous mutants.

[Table 8]

From the above field test results, it was confirmed that K418DM can be usefully used as a PRRSV sadox vaccine in most pig farms in Korea.

Korea Microorganism Conservation Center (overseas) KCCM11458P 20131011

<110> Konkuk University Industrial Cooperation Corp <120> Chimeric strain of Porcine Reproductive and Respiratory Syndrome          Virus <130> P13-095-REA-KKU <150> KR 10-2013-0124958 <151> 2013-10-19 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 19231 <212> DNA <213> Artificial Sequence <220> <223> PRRS K418 <400> 1 atgacgtata ggtgttggct ctatgccatg acatttgtat tgtcaggagc tgcgaccatt 60 ggtacagccc aaaactagct gcacagaaaa cgcccttctg tgacagccct cttcagggga 120 gcttaggggt ctgtccctag caccttgctt ccggagttgc actgctttac ggtctctcca 180 accctttaac catgtctggg atacttgatc ggtgcacgtg cacccccaat gccagggtgt 240 ttatggcgga gggccaagtc tactgcacac gatgtctcag tgcacggtct ctccttcctc 300 tgaatctcca agttcctgag cttggagtgc tgggcctatt ttacaggccc gaagagccac 360 tccggtggac gttgccacgt gcattcccca ctgttgagtg ctcccccgcc ggggcctgct 420 ggctttctgc gatctttcca attgcacgaa tgaccagtgg aaacctgaac tttcaacaaa 480 gaatggtgcg ggtcgcagct gagatttaca gagccggcca gctcacccct gcagtcttga 540 aggctctaca agtttatgaa cggggttgcc gctggtaccc tatagtcgga cctgtccctg 600 gagtggccgt ttttgccaac tccctacatg tgagtgataa acctttcccg ggagcaactc 660 atgtgctaac caacctgcca ctcccgcaga ggcctaagcc tgaagacttt tgcccttttg 720 agtgtgctat ggctgacgtc tatgatattg gtcatggcgc cgtcatgtat gtggccaaag 780 ggaaagtctc ctgggcccct cgtggcgggg atgaggcgaa atttgaaact gtccctaggg 840 agttgaagtt gatcgcgaac caactccaca tctccttccc gccccaccac gcagtggaca 900 tgtctaagtt tgtgttcata gcccctggga gtggtgtctc tatgcgggtc gagtgcccac 960 acggctgtct ccccgctaat actgtccctg aaggtaactg ctggtggcgc ttgtttgact 1020 cgctcccact ggacgttcag aacaaagaaa ttcgccgtgc caaccaattc ggctatcaaa 1080 ccaagcatgg tgtcgctggc aagtacctac aacggaggct gcaagctaat ggtctccgag 1140 cagtgactga tacagatggg cctatcgttg tacagtactt ctctgttagg gagagctgga 1200 tccgccactt cagactggcg gaagagccta gcctccctgg gtttgaagac ctcctcagaa 1260 taagggtaga gcccaatacg tcgccattga gtgacaaggg tggaaaaatc ttccggtttg 1320 gcagtcacaa atggtacggt gctggaaaga gagcaaggaa agcacgctct ggtatgacca 1380 ccacagtcgc tcaccgcgcc ttgcccgctc gtgaaatcca gcaagccaaa aagcacgagg 1440 atgccggcgc tgataaggct gtgcatctca ggcactattc tccgcctgcc gacgggaact 1500 gtggttggca ctgcatttcc gccatcgcca accgaatggt gaattccaaa tttgaaacta 1560 ctcttcccga gagggtgaga ccttcggatg actgggctac tgacgaggac cttgtgaaca 1620 ccatccaaat tctcaagctc cctgcggcct tggacaggaa cggtgcttgt gttggcgcca 1680 aatacgtgct taagttggaa ggcgagcatt ggactgtctc tgtgaccctt gggatgtccc 1740 cttctttgct cccccttgaa tgtgttcagg gctgttgtga gcataagagc ggacttggtc 1800 ccccagatgc ggtcgaagtt ttcggatttg accctgcctg ccttgaccga ctggctgagg 1860 gt; actgtccggc ttccccggtc actactgtgt ggactgtttc acaattcttt gcccgccaca 1980 gaggaggaga gcaccctgat caggtgcgct taggaaaaat catcagcctt tgtcaagttg 2040 ttgaggaatg ctgttgccat cagaataaaa ccaaccgggc caccccggaa gaggttgcgg 2100 caaggattga tcagtacctc catggtgcaa caagtcttga agaatgcttg attaggcttg 2160 agagggtttg cccgccgagc gctgcggaca ccttctttga ttggaatgtt gtgctccctg 2220 gggttggggc ttcaactcag acaaccaaac agctccatgt caaccagtgc cgcgctctgg 2280 ttcctgtcgt gactcaagag cctttggaca aagactcagt ccctctgacc gccttctcgc 2340 tgtccaattg ctactatcct gcacaaggtg acgaggttcg tcaccgtgag aggctaaact 2400 ccgtactctc taagctggag ggggttgttc gtgaggaata tgggctcacg ccaactgaac 2460 ctggcccgcg acccgcacta ccgaacgggc tcgtcgaact taaagaccag atggaggagg 2520 atctgctgaa actagtcaac gcccaggcaa cttcagaaat gatggcctgg gcagccgagc 2580 aggttgatct gaaagcttgg gtcaaaaact acccacggtg gacaccgcca ccccctccac 2640 caagagttca gcctcgaaaa acaaagtctg tcaagagctt gccagggaac aaacctgtcc 2700 ccgctccacg caggaaggtc agatctgatt gtggcagccc gattttgatg ggcgacaatg 2760 ttcctgacgg tcgggaagat ttgactgttg gtggccccct tgatctttcg acaccatccg 2820 agccgatgac acctctgagt gagcctgcac ttatgcccgc gttgcaatat atttctaggc 2880 cagtgacatc tttgagtgtg ctggccccag ttcctgcacc gcgtagaact gtgtcccgac 2940 cggtgacgcc cttgagtgag ccaatttttg tgtctgcacc gcgacacaaa tttcagcagg 3000 tggaagaagc gaatctggcg gcaacaacgc tgacgcacca ggacgaacct ctagatttgt 3060 ctgcatcctc acagactgaa tatgaggctt ctcccctaac accactgcag aacatgggta 3120 ttctggaggt gggggggcaa gaagctgagg aagttctgag tgaaatctcg gatacactga 3180 atgacatcaa ccctgcacct gtgtcatcaa gcagctccct gtcaagtgtt aagatcacac 3240 gcccaaaaca ctctgctcaa gccatcattg actcgggcgg gccctgcagt gggcatctcc 3300 gaagggaaaa agaagcatgc ctcagcatca tgcgtgaggc ttgtgatgcg gctaagctta 3360 gtgaccctgc cacgcaggaa tggctttctc gcatgtggga tagggttgac atgctgactt 3420 ggcgcaacac gtctgcttac caggcgttcc gcatcttaga tggtaggttt gagtttctcc 3480 caaagatgat actcgagaca ccgccgccct acccgtgtgg gtttgtgatg ctgcctcaca 3540 cgcctgcacc ttccgtgggt gcagagagtg accttaccat tggttcagtc gccactgaag 3600 atgttccacg catcctcggg aaaatagaaa acgccggcga gatgcccaac caggggctct 3660 tgacatcctt cggggaagaa ccggtgtgcg accaacctgt caaggactcc tggatgtcgt 3720 cgcgggggtt tgacgagagc acaacggctc cgtccgctgg tacaggtggt gctgacttac 3780 ccaccgattt gccaccttca gatggtttgg atgcggacga gtgggggccg ttacggacgg 3840 taagaaagaa agctgaaagg ctcttcgacc aattgagccg tcaggttttt aacctcgtct 3900 cccatctccc tgttttcttc tcacacctct tcaaatctga cagtggttat tctccgggtg 3960 attggggttt tgcagctttt actttatttt gcctcttttt gtgttacagc tacccattct 4020 ttggttttgt tcccctcttg ggtgtttttt ctgggtcttc tcggcgtgtg cgcatggggg 4080 tttttggctg ttggttggct tttgctgttg gcctgttcaa gcctgtgtcc gacccagtcg 4140 gcactgcttg tgagtttgac tcgccagagt gtaggaacgt ccttcattct tttgagcttc 4200 tcaaaccttg ggaccctgtt cgcagccttg ttgtgggccc cgtcggtctc ggccttgcca 4260 ttcttggcag gttactgggc ggggcacgct acatctggca ttttttgctt aggcttggca 4320 ttgttgcaga ttgtatcttg gctggagctt atgtgctttc tcaaggtagg tgtaaaaagt 4380 gctggggatc ttgtgtaaga actgctccta atgaaatcgc cttcaacgtg ttccctttta 4440 cgcgtgcgac caggtcgtca ctcatcgacc tgtgcgatcg gttttgtgcg ccaaaaggca 4500 tggaccccat tttcctcgct actgggtggc gcgggtgctg gaccggccga agtcccattg 4560 agcaaccctc tgaaaaaccc atcgcgttcg cccagttgga tgaaaagagg attacggcta 4620 gaactgtggt cgctcagcct tatgatccta accaagccgt aaagtgcttg cgggtgttac 4680 aggcgggtgg ggcgatagtg gccgaggcag tcccaaaagt ggtcaaggtt tccgctattc 4740 cattccgagc tccctttttt cccaccggag tgaaggttga tcctgagtgc aggatcgtgg 4800 tcgaccccga cacttttact acagctctcc ggtctggtta ctccaccaca aacctcgtcc 4860 ttggtgtggg ggactttgcc caactgaatg gattaaaaat caggcaaatt tccaagccct 4920 cgggaggagg cccgcacctc attgctgccc tgcatgttgc ttgctcgatg gcgttgcaca 4980 tgcttgctgg agtttatgta actgcagtgg ggtcttgcgg taccggcacc aacgatccgt 5040 ggtgcactaa cccattcgcc gtccctggct acggacctgg ctccctctgc acgtccagat 5100 tgtgcatctc ccaacatggc cttaccctgc ccttgacagc acttgtggca ggattcggtc 5160 ttcaggaaat tgccctagtc gttttgattt tcgtttccat cggaggcatg gctcataggt 5220 tgagttgtaa ggctgatatg ctgtgcgtct tacttgcaat cgccagctat gtttgggtac 5280 cccttacctg gttgctctgt gtgtttcctt gctggttgcg ctggttctct ttgcaccctc 5340 tcaccattct atggttggtg tttttcttga tgtctgtaaa tatgccttcg ggaatcttaa 5400 ccgtggtgtt attggttgct ctttggcttc taggccgtta tactaatgtt gttggtcttg 5460 ttacccccta tgatattcat cattacacca atggcccccg cggtgttgcc gccttggcta 5520 ccgcaccaga tgggacttac ttggccgctg tccgccgcgc tgcgttgact ggccgcaccg 5580 tgctgtttac cccgtctcag cttgggtccc ttcttgaggg cgctttcaga actcgaaagc 5640 cctcactgaa caccgtcaat gtggtcgggt cctccatggg ctctggcgga gtgttcacta 5700 tcgatgggaa aattaagtgc gtgactgccg cacatgtcct tacgggtaat tcagctaggg 5760 tttccggggt cggcttcaat caaatgcttg actttgatgt aaaaggggac ttcgccatag 5820 ctgattgccc gaattggcaa ggggctgctc ctaagaccca attctgcgag gatggatgga 5880 ctggccgcgc ctattggctg acatcctctg gcgtcgaacc cggtgtcatt gggaatggat 5940 tcgccttctg cttcaccgcg tgcggcgatt ccgggtcccc agtgatcacc gaagccggtg 6000 agcttgtcgg cgttcacaca ggatcaaaca aacaaggagg aggcattgtt acgcgcccct 6060 ctggccagtt ttgcaatgtg gcacccatca agctgagcga attaagtgag ttctttgctg 6120 gacctaaggt cccgctcggt gatgtgaagg ttggcagcca cataattaaa gacatatgcg 6180 aggtaccttc agatctttgc gccttgcttg ctgccaaacc cgaactggaa ggaggcctct 6240 ccaccgtcca acttctgtgt gtgtttttcc tcctgtggag aatgatggga catgcctgga 6300 cgcccttggt tgctgttggg ttttttatct tgaatgaggt tctcccagct gtactggtcc 6360 ggagtgtttt ctcctttgga atgtttgtgc tatcttggct cacaccatgg tctgcgcaag 6420 ttctgatgat caggcttcta acagcagctc ttaacaggaa cagattgtca ctcgcctttt 6480 acagccttgg tgcagcgacc ggttttgtcg cagatctggc ggcaactcaa gggcacccgt 6540 tgcaggcagt aatgaattta agtacctatg ccttcctgcc tcggataatg gtcgtgacct 6600 caccagtccc agtgattgcg tgtggtgttg tgcacctcct tgccataatt ttgtacttgt 6660 ttaagtaccg ctgcctgcac aatgtccttg ttggcgatgg tgcgttctct gcggctttct 6720 tcttgcgata ctttgccgag gggaaattga gggaaggggt gtcgcaatcc tgcgggatga 6780 atcatgagtc gctgactggt gccctcgcta tgagacttaa tgacgaggac ttggattttc 6840 ttacgaaatg gactgatttt aagtgttttg tttctgcatc caacatgagg aatgcggcgg 6900 gccagttcat cgaggctgcc tatgctaaag cacttagaat tgaacttgcc cagttggtgc 6960 aggttgataa ggttcgaggt actttggcca aacttgaagc ttttgctgat accgtggcac 7020 cccaactctc gcccggtgac attgttgttg ctcttggcca tacgcctgtt ggcggtatct 7080 tcgacctaaa ggttggtagc accaagcata ccctccaagc cattgagacc agagttcttg 7140 ccgggtccaa aatgaccgtg gcgcgtgtcg ttgatccaac ccccacaccc ccacccgcac 7200 ccgtgcctat cccccttcca ccgaaagttc tggagaatgg tcccaacgcc tggggggatg 7260 aggatcgttt gaataagaag aagaggcgca ggatggaagc cgtcggcatc tttgttatgg 7320 gtggaaagaa atatcagaaa ttttgggaca agaactccgg tgatgtgttt tatgaggagg 7380 tccatgataa cacagacgcg tgggagtgcc tcagagttga caaccctgcc gactttgacc 7440 ctgagaaggg aactctgtgc gggcatacta ccattgaaga taagacttac agtgtctacg 7500 cctccccatc tggcaagaaa ttcctggtcc ccgtctaccc agagagcaaa aaaaaccaat 7560 gggaagctgc gaagctttcc gtggaacagg cccttggcat gatgaatgtc gacggtgaac 7620 tgacagccaa agaagtggag aaactgaaaa gaataattga caaactccag ggcctgacta 7680 aggagcagtg tttaaactgc tagccgccag cggcttgacc cgctgtggtc gcggcggctt 7740 ggttgttact gagacycgg taaaaatagt caaatttcac aaccggacct tcaccctagg 7800 acctgtgaat ttaaaagtgg ccagtgaggt tgagctaaaa gacgcggtcg agcataacca 7860 acacccggtt gcaagaccgg ttgatggtgg tgttgtgctc ctgcgctccg cagttccttc 7920 gcttatagac gtcttaatct ccggcgctga tgcatctccc aagttactcg cccgccacgg 7980 gccgggaaac actgggatcg atggcacgct ttgggatttt gaggccgagg ccactaaaga 8040 ggaaattgca ctcagtgcgc aaataataca ggcttgtgac attaggcgcg gcgacgcacc 8100 tgaaattggt cttccttata agctgtaccc tgtcaggggc aaccctgagc gggtaaaagg 8160 agttttacag aatacaaggt ttggagacat accttataaa acccccagtg acactggaag 8220 cccagtgcac gcggctgcct gcctcacgcc caatgccact ccggtgactg atgggcgctc 8280 cgtcttggcc acgactatgc cctccggttt tgagttgtat gtaccgacca ttccagcgtc 8340 tgtccttgat tatcttgatt ctaggcctga ctgccccaaa cagttgacag agcacggctg 8400 tgaggacgcc gcattaagag acctctccaa gtatgacttg tccacccaag gctttgtttt 8460 acctggagtt cttcgccttg tgcgtaagta cctgtttgct catgtgggta agtgcccgcc 8520 cgttcatcgg ccttccactt accctgccaa gaattctatg gctggaataa atgggaacag 8580 gtttccacc aaggacatcc agagcgtccc tgaaatcgac gttctgtgcg cacaggccgt 8640 gcgggaaaac tggcaaactg ttaccccttg taccctcaag aaacagtatt gtgggaagaa 8700 gaagactagg acaatactcg gcaccaataa cttcattgcg ctggcccacc gggcagcgtt 8760 gagtggtgtc acccagggct tcatgaaaaa ggcgtttaac tcgcccattg ccctcggtaa 8820 aaacaaattt aaagagcttc agactccggt cttaggcagg tgccttgaag ctgatcttgc 8880 atcctgcgat cgctccacac ctgcaattgt ccgctggttt gccgccaatc ttctttatga 8940 acttgcctgt gctgaagagc acctgccgtc gtacgtgttg aactgctgcc acgacctact 9000 ggtcacgcag tccggcgcag taactaagag aggtggcctg tcgtctggcg acccgatcac 9060 ttctgtgtcc aacaccattt acagcttggt gatatatgca caacacatgg tgctcagtta 9120 ctttaaaagt ggtcaccctc atggccttct gtttctacaa gaccagctga agtttgagga 9180 catgctcaag gttcaacccc tgatcgtcta ttcggacgac ctcgtactgt atgccgagtc 9240 tcccaccatg ccaaactacc actggtgggt tgaacatctg aacctgatgc tgggttttca 9300 gacggaccca aagaagacag ccataacaga ctcgccatca tttctaggct gtaggataat 9360 aaatggacgc cagctcgtcc ctaaccgtga caggattctc gcggccctcg cctaccatat 9420 gaaggcaagc aatgtctctg aatactacgc ctcggcggct gcgatactca tggacagctg 9480 tgcttgttta gagtatgatc ccgaatggtt tgaagagctt gtagttggga tagcgcagtg 9540 tgcccgcaag gacggctaca gttttcccgg cccgccgttc ttcttgtcca tgtgggaaaa 9600 actcagatcc aatcatgagg ggaagaagtc cagaatgtgc gggtactgcg gggccccggc 9660 tccgtacgcc actgcctgtg gcctcgacgt ctgtatttac cacacccact tccaccagca 9720 ttgtccagtc atcatctggt gtggccaccc ggctggttct ggttcttgta gtgagtgcaa 9780 acccccccta gggaaaggca caagccctct agatgaggtg ttggaacaag tcccgtataa 9840 gcctccacgg actgtaatca tgcatgtgga gcagggtctc acccctcttg acccaggcag 9900 ataccagact cgccgcggat tagtctccgt taggcgtggc attagaggaa atgaggttga 9960 tctaccagac ggtgattatg ctagcaccgc cctactccct acttgtaaag agattaacat 10020 ggtcgctgtc gcctctaatg tgttgcgcag caggttcatc atcggcccgc ctggtgctgg 10080 gaaaacatac tggctccttc aacaggtcca ggatggtgat gtcatttaca cgccaactca 10140 ccagaccatg ctcgacatga ttagggcttt ggggacgtgc cggttcaacg tcccagcagg 10200 tacgacgctg caattccctg ccccctcccg taccggccct tgggttcgca tcctagccgg 10260 cggttggtgt cctggcaaga attccttcct ggatgaagca gcgtattgta atcaccttga 10320 tgtcttgagg cttcttagca aaactaccct cacctgtctg ggagatttca aacaactcca 10380 cccagtgggt tttgattctc attgctatgt ttttgacatc atgcctcaga ctcaactgaa 10440 gaccatctgg agatttggac agaatatctg tgatgccatt cagccagatt acagggacaa 10500 acttgtatcc atggtcaaca caacccgtgt aacctacgtg gaaaaacctg tcaagtatgg 10560 gcaagtcctc accccttacc acagggaccg agaggacggc gccatcacaa ttgactccag 10620 tcaaggcgcc acatttgatg tggttacact gcatttgccc actaaagatt cactcaacag 10680 gcaaagagcc cttgttgcta ttaccagggc aagacatgct atctttgtgt atgacccaca 10740 caggcaactg cagagcatgt ttgatcttcc tgcgaaaggc acacccgtca acctcgctgt 10800 gcaccgtgac gagcagctga tcgtgctaga tagaaataac aaagaatgca cggttgctca 10860 ggctctaggc aatggggata aattcagggc cacagacaag cgcgttgtag attctctccg 10920 cgccatttgt gcagatctgg aagggtcgag ctccccgctc cccaaggtcg cacacaactt 10980 gggattttat ttctcgcctg atttgacaca gtttgctaaa ctcccggtag aacttgcacc 11040 ccactggccc gtggtgacaa cccagaacaa tgaaaagtgg ccagaccggt tggttgctag 11100 ccttcgcccc gtccataagt atagccgcgc gtgcatcggt gccggctaca tggtgggccc 11160 ctcagtgttt ctgggcaccc ctggggttgt gtcatactat ctcacaaaat ttgtcagggg 11220 cgaggctcaa atgcttccgg agacagtctt cagcaccggc cgaattgagg tagattgccg 11280 tgagtatctt gatgaccggg agcgagaaat tgctgagtcc ctcccccatg ctttcattgg 11340 cgacgtcaaa ggcactaccg ttggaggatg tcaccatgtc acctccaaat accttccgcg 11400 cttccttccc aaggaatcag tcgcggtagt cggggtttca agccccggga aagccgcaaa 11460 agcagtttgc acattaacag atgtgtatct cccagatctc gaagcttacc tccacccaga 11520 gacccagtcc aagtgctgga aaatgatgtt ggacttcaag gaagttcgac tgatggtctg 11580 gaaggacaag acggcctatt ttcaacttga aggccgccat ttcacctggt accagcttgc 11640 aagctatgcc tcgtacatcc gagttcctgt taactctacg gtgtatttgg acccctgcat 11700 gggccctgcc ctttgcaaca gaagagttgt cgggtccact cattggggag ctgacctcgc 11760 agtcacccct tatgattacg gtgccaaaat catcctgtct agtgcatacc atggtgaaat 11820 gccccctggg tacaaaatcc tggcgtgcgc ggagttctcg cttgacgatc cagtgaggta 11880 caaacacacc tgggggtttg aatcggatac agcgtatctg tacgagttca ccggaaacgg 11940 tgaggactgg gaggattaca atgatgcgtt tcgtgcgcgc cagaaaggga aaatttataa 12000 ggccactgcc accagcatga ggtttcattt tcccccgggc cctgtcattg aaccaacttt 12060 aggcctgaat tgaaatgaaa tggggtccat gcaaagcctc tttgacaaaa ttggccaact 12120 ttttgtggat gctttcacgg aatttttggt gtccattgtt gatatcatta tatttttggc 12180 cattttgttt ggcttcacca tcgccggttg gctggtggtc ttttgcatca gattggtttg 12240 ctccgcgata ctccgtgcgc gctctaccat tcactctgag caattacaga agatcctatg 12300 aggccttcct ctcccagtgt caagtggaca tccccgcttg gggaaccaaa catcccctag 12360 ggatgctttg gcaccacaag gtatcgaccc tgatcgatga aatggtgtcg cgtcgaatgt 12420 accgcatcat ggaaaaggca ggacaggctg cctggaaaca ggtggtgacc gaggccacgc 12480 tgtctcggat tagtagcttg gatgtggtag ctcattttca gcaccttgcc gccattgaag 12540 ccgagacctg taaatattta gcatcccggc tgcccatgct acacaacctg cgcttgacag 12600 ggtcaaatgt aaccatagtg tataatagta ctttgaatca ggtgtttgct gtttttccaa 12660 cccccggttc ccggccaaag cttcatgatt ttcagcaatg gttaatagct gtacattcct 12720 ccatattttc ctctgttgca gcttcttgta ctctttttgt tgtgctgtgg ttgcggattc 12780 caatgctacg tactgctttt ggtttccgct ggttaggggc aatttttctt tcgaactcac 12840 agtgaattac acggtgtgcc cgccttgcct cacccgacag gcggccgcag agatctacga 12900 acccggtagg tctctttggt gtaggatagg gcacgatcga tgtggggagg atgaccacga 12960 cgagttaggg ttcatggtac cacctggcct ctccaacgaa ggtcatttgg ccagtgttta 13020 cgcctggttg gcgttcttgt cctttagcta cacagcccag ttccaccctg aaatatttgg 13080 gatagggaat gtgagcagag tttatgttga catcaagcat caattcatct gcgctgttca 13140 tgacggacag aacaccacct tgcctcgcca cgacaacatc tcagctgtgt tccagaccta 13200 ttaccaacat caggtcgacg gcggcaattg gtttcaccta gaatggttgc gtcccttctt 13260 ttcctcttgg ttggttttga acgtctcttg gtttctcagg cgttcgcctg caagccatgt 13320 ttcagttcga gtctttcaga cattaagacc aacaccaccg cggcggcagg ctttgctatc 13380 ctccaggaca tcagctgtct taggcatggc aactcaacct ctgaggcgtt tcgcaagatt 13440 cctcaatgcc gcacggcgat agggacacca gtgtatatca ccatcacagc caatgtaaca 13500 gatgagaatt atttacattc ttctgacctt ctcatgctct ctgcttgcct tttctatgct 13560 tctgagatga gcgaaaaggg atttaaggtg gtatttggca atgtgtcagg catcgtggct 13620 gtgtgtgtca attttaccag ctacgtccaa cacgtcaagg agtctaccca gcgctcctta 13680 gtggtcaatc atgtacggct gctccatttc atgacacctg agaccatgag gtgggcgact 13740 gttttagcct gtctttttgc cattctgttg gccatttgaa tgtttaagta tgttggggag 13800 atgcttgacc gcgggctgtt gctcgcaatt gctttttttg tggtgtatcg tgccatcttg 13860 ttttgttgcg atcgtcagcg ccaacaacag cagcagctca aatttacagc tgatttacaa 13920 cttgacgcta tgtgagctga atggcacaga ttggctagct aacagatttg actgggcggt 13980 ggagtgtttt gttatttttc ctgtattgac tcacattgtc tcttatggtg ccctaaccac 14040 tagccacttc cttgacacag tcggtttggt cactgtgtct accgccggat ttgttcacgg 14100 gcggtatgtt ttgagtagca tttacgcggt ctgtgccctg gctgcgttga tttgcttcgt 14160 cattaggctt gcgaagaatt gcatgtcctg gcgctactca tgtaccagat ataccaactt 14220 tcttctagat accaagggca gactctaccg ttggcggtcg cctgtcatta tagagaaaag 14280 gggcaaagtt gaggtcgagg gtaaaaaaat cgacctcaaa agagttgtgc ttgatggttc 14340 cgcggcaacc cctgtaacca gagtttcggc ggagcaatgg ggtcatcctt agatgacttc 14400 tgccatgata gcacagcccc acaaaaggtg ctcttggcgt tttctatcac ctacacgcca 14460 gtgatgatat acgccctaaa ggtaagtcgc ggccgactgc tagggctttt gcaccttttg 14520 atcttcctga attgtgcttt caccttcggg tatatgacat tcatgcactt tcagagtaca 14580 aataaggtcg cgctcactat gggagcagta gtggcactcc tttggggggt gtactcagcc 14640 atagaaacct ggagattcat cacctccaga tgccgtttgt gcttgctagg ccgcaagtac 14700 attctggccc ctgcccacca cgttgaaagt gccgcaggct ttcatccgat tgcggcaaat 14760 gataaccacg catttgtcgt ccggcgtccc ggctctacta cggtcaacgg cacattggtg 14820 cccgggttga aaagcctcgt gttgggtggc agaaaagctg ttaaacaggg agtggtaaac 14880 cttgttaaat atgccaaata acaacggcaa gcagcagaag agaaagaagg gggatggcca 14940 gccagtcaat cagctgtgcc aaatgctggg caaaattatc gcccagcaaa accagtccag 15000 aggcaaggga ccgggaaaga aaaataagaa gaaaaacccc gagaagcccc atttccctct 15060 tgcggctgaa gatgatgtca gacatcactt tacccctagt gagcggcaat tgtgtctgtc 15120 gtcaatccag accgccttta atcaaggcgc tgggacctgc accctatcag attcagggag 15180 gataagttac actgtggagt ttagtttgcc tacgcatcat actgtgcgcc tgatccgtgt 15240 cacagcatca ccttcagcat gatgagctgg cattcttgtg gcatcccagt atttgaattg 15300 gaataatgcg tggtgaatgg cactgattga cattgtgcct tcaagtcacc tattcaatta 15360 gggcgaccgt gtgggggcaa gatttaattg gcgagaacca cacggccgaa attaaaaaaa 15420 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaacgtt aattaacgat gcccttgaga 15480 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 15540 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt 15600 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc 15660 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc 15720 gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg 15780 ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc cggcggcatc 15840 gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag 15900 cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc gctgatcgtc 15960 acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc 16020 gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg 16080 acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga attggagcca 16140 atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg 16200 cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg tcctggccac 16260 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta 16320 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa 16380 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg 16440 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct 16500 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga 16560 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta 16620 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta 16680 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa 16740 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac 16800 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg 16860 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 16920 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 16980 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 17040 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt 17100 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 17160 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 17220 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 17280 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 17340 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 17400 gtgggagaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 17460 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 17520 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 17580 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 17640 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 17700 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 17760 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 17820 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 17880 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 17940 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 18000 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 18060 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 18120 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 18180 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 18240 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 18300 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 18360 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc 18420 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 18480 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 18540 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 18600 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 18660 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac 18720 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 18780 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 18840 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 18900 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 18960 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 19020 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 19080 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 19140 gcgtatcacg aggccctttc gtcttcaaga attctcatgt ttgacagctt atcatcgata 19200 agcttcggac cgtaatacga ctcactatag g 19231 <210> 2 <211> 19231 <212> DNA <213> Artificial Sequence <220> <223> PRRS K418DM <400> 2 atgacgtata ggtgttggct ctatgccatg acatttgtat tgtcaggagc tgcgaccatt 60 ggtacagccc aaaactagct gcacagaaaa cgcccttctg tgacagccct cttcagggga 120 gcttaggggt ctgtccctag caccttgctt ccggagttgc actgctttac ggtctctcca 180 accctttaac catgtctggg atacttgatc ggtgcacgtg cacccccaat gccagggtgt 240 ttatggcgga gggccaagtc tactgcacac gatgtctcag tgcacggtct ctccttcctc 300 tgaatctcca agttcctgag cttggagtgc tgggcctatt ttacaggccc gaagagccac 360 tccggtggac gttgccacgt gcattcccca ctgttgagtg ctcccccgcc ggggcctgct 420 ggctttctgc gatctttcca attgcacgaa tgaccagtgg aaacctgaac tttcaacaaa 480 gaatggtgcg ggtcgcagct gagatttaca gagccggcca gctcacccct gcagtcttga 540 aggctctaca agtttatgaa cggggttgcc gctggtaccc tatagtcgga cctgtccctg 600 gagtggccgt ttttgccaac tccctacatg tgagtgataa acctttcccg ggagcaactc 660 atgtgctaac caacctgcca ctcccgcaga ggcctaagcc tgaagacttt tgcccttttg 720 agtgtgctat ggctgacgtc tatgatattg gtcatggcgc cgtcatgtat gtggccaaag 780 ggaaagtctc ctgggcccct cgtggcgggg atgaggcgaa atttgaaact gtccctaggg 840 agttgaagtt gatcgcgaac caactccaca tctccttccc gccccaccac gcagtggaca 900 tgtctaagtt tgtgttcata gcccctggga gtggtgtctc tatgcgggtc gagtgcccac 960 acggctgtct ccccgctaat actgtccctg aaggtaactg ctggtggcgc ttgtttgact 1020 cgctcccact ggacgttcag aacaaagaaa ttcgccgtgc caaccaattc ggctatcaaa 1080 ccaagcatgg tgtcgctggc aagtacctac aacggaggct gcaagctaat ggtctccgag 1140 cagtgactga tacagatggg cctatcgttg tacagtactt ctctgttagg gagagctgga 1200 tccgccactt cagactggcg gaagagccta gcctccctgg gtttgaagac ctcctcagaa 1260 taagggtaga gcccaatacg tcgccattga gtgacaaggg tggaaaaatc ttccggtttg 1320 gcagtcacaa atggtacggt gctggaaaga gagcaaggaa agcacgctct ggtatgacca 1380 ccacagtcgc tcaccgcgcc ttgcccgctc gtgaaatcca gcaagccaaa aagcacgagg 1440 atgccggcgc tgataaggct gtgcatctca ggcactattc tccgcctgcc gacgggaact 1500 gtggttggca ctgcatttcc gccatcgcca accgaatggt gaattccaaa tttgaaacta 1560 ctcttcccga gagggtgaga ccttcggatg actgggctac tgacgaggac cttgtgaaca 1620 ccatccaaat tctcaagctc cctgcggcct tggacaggaa cggtgcttgt gttggcgcca 1680 aatacgtgct taagttggaa ggcgagcatt ggactgtctc tgtgaccctt gggatgtccc 1740 cttctttgct cccccttgaa tgtgttcagg gctgttgtga gcataagagc ggacttggtc 1800 ccccagatgc ggtcgaagtt ttcggatttg accctgcctg ccttgaccga ctggctgagg 1860 gt; actgtccggc ttccccggtc actactgtgt ggactgtttc acaattcttt gcccgccaca 1980 gaggaggaga gcaccctgat caggtgcgct taggaaaaat catcagcctt tgtcaagttg 2040 ttgaggaatg ctgttgccat cagaataaaa ccaaccgggc caccccggaa gaggttgcgg 2100 caaggattga tcagtacctc catggtgcaa caagtcttga agaatgcttg attaggcttg 2160 agagggtttg cccgccgagc gctgcggaca ccttctttga ttggaatgtt gtgctccctg 2220 gggttggggc ttcaactcag acaaccaaac agctccatgt caaccagtgc cgcgctctgg 2280 ttcctgtcgt gactcaagag cctttggaca aagactcagt ccctctgacc gccttctcgc 2340 tgtccaattg ctactatcct gcacaaggtg acgaggttcg tcaccgtgag aggctaaact 2400 ccgtactctc taagctggag ggggttgttc gtgaggaata tgggctcacg ccaactgaac 2460 ctggcccgcg acccgcacta ccgaacgggc tcgtcgaact taaagaccag atggaggagg 2520 atctgctgaa actagtcaac gcccaggcaa cttcagaaat gatggcctgg gcagccgagc 2580 aggttgatct gaaagcttgg gtcaaaaact acccacggtg gacaccgcca ccccctccac 2640 caagagttca gcctcgaaaa acaaagtctg tcaagagctt gccagggaac aaacctgtcc 2700 ccgctccacg caggaaggtc agatctgatt gtggcagccc gattttgatg ggcgacaatg 2760 ttcctgacgg tcgggaagat ttgactgttg gtggccccct tgatctttcg acaccatccg 2820 agccgatgac acctctgagt gagcctgcac ttatgcccgc gttgcaatat atttctaggc 2880 cagtgacatc tttgagtgtg ctggccccag ttcctgcacc gcgtagaact gtgtcccgac 2940 cggtgacgcc cttgagtgag ccaatttttg tgtctgcacc gcgacacaaa tttcagcagg 3000 tggaagaagc gaatctggcg gcaacaacgc tgacgcacca ggacgaacct ctagatttgt 3060 ctgcatcctc acagactgaa tatgaggctt ctcccctaac accactgcag aacatgggta 3120 ttctggaggt gggggggcaa gaagctgagg aagttctgag tgaaatctcg gatacactga 3180 atgacatcaa ccctgcacct gtgtcatcaa gcagctccct gtcaagtgtt aagatcacac 3240 gcccaaaaca ctctgctcaa gccatcattg actcgggcgg gccctgcagt gggcatctcc 3300 gaagggaaaa agaagcatgc ctcagcatca tgcgtgaggc ttgtgatgcg gctaagctta 3360 gtgaccctgc cacgcaggaa tggctttctc gcatgtggga tagggttgac atgctgactt 3420 ggcgcaacac gtctgcttac caggcgttcc gcatcttaga tggtaggttt gagtttctcc 3480 caaagatgat actcgagaca ccgccgccct acccgtgtgg gtttgtgatg ctgcctcaca 3540 cgcctgcacc ttccgtgggt gcagagagtg accttaccat tggttcagtc gccactgaag 3600 atgttccacg catcctcggg aaaatagaaa acgccggcga gatgcccaac caggggctct 3660 tgacatcctt cggggaagaa ccggtgtgcg accaacctgt caaggactcc tggatgtcgt 3720 cgcgggggtt tgacgagagc acaacggctc cgtccgctgg tacaggtggt gctgacttac 3780 ccaccgattt gccaccttca gatggtttgg atgcggacga gtgggggccg ttacggacgg 3840 taagaaagaa agctgaaagg ctcttcgacc aattgagccg tcaggttttt aacctcgtct 3900 cccatctccc tgttttcttc tcacacctct tcaaatctga cagtggttat tctccgggtg 3960 attggggttt tgcagctttt actttatttt gcctcttttt gtgttacagc tacccattct 4020 ttggttttgt tcccctcttg ggtgtttttt ctgggtcttc tcggcgtgtg cgcatggggg 4080 tttttggctg ttggttggct tttgctgttg gcctgttcaa gcctgtgtcc gacccagtcg 4140 gcactgcttg tgagtttgac tcgccagagt gtaggaacgt ccttcattct tttgagcttc 4200 tcaaaccttg ggaccctgtt cgcagccttg ttgtgggccc cgtcggtctc ggccttgcca 4260 ttcttggcag gttactgggc ggggcacgct acatctggca ttttttgctt aggcttggca 4320 ttgttgcaga ttgtatcttg gctggagctt atgtgctttc tcaaggtagg tgtaaaaagt 4380 gctggggatc ttgtgtaaga actgctccta atgaaatcgc cttcaacgtg ttccctttta 4440 cgcgtgcgac caggtcgtca ctcatcgacc tgtgcgatcg gttttgtgcg ccaaaaggca 4500 tggaccccat tttcctcgct actgggtggc gcgggtgctg gaccggccga agtcccattg 4560 agcaaccctc tgaaaaaccc atcgcgttcg cccagttgga tgaaaagagg attacggcta 4620 gaactgtggt cgctcagcct tatgatccta accaagccgt aaagtgcttg cgggtgttac 4680 aggcgggtgg ggcgatagtg gccgaggcag tcccaaaagt ggtcaaggtt tccgctattc 4740 cattccgagc tccctttttt cccaccggag tgaaggttga tcctgagtgc aggatcgtgg 4800 tcgaccccga cacttttact acagctctcc ggtctggtta ctccaccaca aacctcgtcc 4860 ttggtgtggg ggactttgcc caactgaatg gattaaaaat caggcaaatt tccaagccct 4920 cgggaggagg cccgcacctc attgctgccc tgcatgttgc ttgctcgatg gcgttgcaca 4980 tgcttgctgg agtttatgta actgcagtgg ggtcttgcgg taccggcacc aacgatccgt 5040 ggtgcactaa cccattcgcc gtccctggct acggacctgg ctccctctgc acgtccagat 5100 tgtgcatctc ccaacatggc cttaccctgc ccttgacagc acttgtggca ggattcggtc 5160 ttcaggaaat tgccctagtc gttttgattt tcgtttccat cggaggcatg gctcataggt 5220 tgagttgtaa ggctgatatg ctgtgcgtct tacttgcaat cgccagctat gtttgggtac 5280 cccttacctg gttgctctgt gtgtttcctt gctggttgcg ctggttctct ttgcaccctc 5340 tcaccattct atggttggtg tttttcttga tgtctgtaaa tatgccttcg ggaatcttaa 5400 ccgtggtgtt attggttgct ctttggcttc taggccgtta tactaatgtt gttggtcttg 5460 ttacccccta tgatattcat cattacacca atggcccccg cggtgttgcc gccttggcta 5520 ccgcaccaga tgggacttac ttggccgctg tccgccgcgc tgcgttgact ggccgcaccg 5580 tgctgtttac cccgtctcag cttgggtccc ttcttgaggg cgctttcaga actcgaaagc 5640 cctcactgaa caccgtcaat gtggtcgggt cctccatggg ctctggcgga gtgttcacta 5700 tcgatgggaa aattaagtgc gtgactgccg cacatgtcct tacgggtaat tcagctaggg 5760 tttccggggt cggcttcaat caaatgcttg actttgatgt aaaaggggac ttcgccatag 5820 ctgattgccc gaattggcaa ggggctgctc ctaagaccca attctgcgag gatggatgga 5880 ctggccgcgc ctattggctg acatcctctg gcgtcgaacc cggtgtcatt gggaatggat 5940 tcgccttctg cttcaccgcg tgcggcgatt ccgggtcccc agtgatcacc gaagccggtg 6000 agcttgtcgg cgttcacaca ggatcaaaca aacaaggagg aggcattgtt acgcgcccct 6060 ctggccagtt ttgcaatgtg gcacccatca agctgagcga attaagtgag ttctttgctg 6120 gacctaaggt cccgctcggt gatgtgaagg ttggcagcca cataattaaa gacatatgcg 6180 aggtaccttc agatctttgc gccttgcttg ctgccaaacc cgaactggaa ggaggcctct 6240 ccaccgtcca acttctgtgt gtgtttttcc tcctgtggag aatgatggga catgcctgga 6300 cgcccttggt tgctgttggg ttttttatct tgaatgaggt tctcccagct gtactggtcc 6360 ggagtgtttt ctcctttgga atgtttgtgc tatcttggct cacaccatgg tctgcgcaag 6420 ttctgatgat caggcttcta acagcagctc ttaacaggaa cagattgtca ctcgcctttt 6480 acagccttgg tgcagcgacc ggttttgtcg cagatctggc ggcaactcaa gggcacccgt 6540 tgcaggcagt aatgaattta agtacctatg ccttcctgcc tcggataatg gtcgtgacct 6600 caccagtccc agtgattgcg tgtggtgttg tgcacctcct tgccataatt ttgtacttgt 6660 ttaagtaccg ctgcctgcac aatgtccttg ttggcgatgg tgcgttctct gcggctttct 6720 tcttgcgata ctttgccgag gggaaattga gggaaggggt gtcgcaatcc tgcgggatga 6780 atcatgagtc gctgactggt gccctcgcta tgagacttaa tgacgaggac ttggattttc 6840 ttacgaaatg gactgatttt aagtgttttg tttctgcatc caacatgagg aatgcggcgg 6900 gccagttcat cgaggctgcc tatgctaaag cacttagaat tgaacttgcc cagttggtgc 6960 aggttgataa ggttcgaggt actttggcca aacttgaagc ttttgctgat accgtggcac 7020 cccaactctc gcccggtgac attgttgttg ctcttggcca tacgcctgtt ggcggtatct 7080 tcgacctaaa ggttggtagc accaagcata ccctccaagc cattgagacc agagttcttg 7140 ccgggtccaa aatgaccgtg gcgcgtgtcg ttgatccaac ccccacaccc ccacccgcac 7200 ccgtgcctat cccccttcca ccgaaagttc tggagaatgg tcccaacgcc tggggggatg 7260 aggatcgttt gaataagaag aagaggcgca ggatggaagc cgtcggcatc tttgttatgg 7320 gtggaaagaa atatcagaaa ttttgggaca agaactccgg tgatgtgttt tatgaggagg 7380 tccatgataa cacagacgcg tgggagtgcc tcagagttga caaccctgcc gactttgacc 7440 ctgagaaggg aactctgtgc gggcatacta ccattgaaga taagacttac agtgtctacg 7500 cctccccatc tggcaagaaa ttcctggtcc ccgtctaccc agagagcaaa aaaaaccaat 7560 gggaagctgc gaagctttcc gtggaacagg cccttggcat gatgaatgtc gacggtgaac 7620 tgacagccaa agaagtggag aaactgaaaa gaataattga caaactccag ggcctgacta 7680 aggagcagtg tttaaactgc tagccgccag cggcttgacc cgctgtggtc gcggcggctt 7740 ggttgttact gagacycgg taaaaatagt caaatttcac aaccggacct tcaccctagg 7800 acctgtgaat ttaaaagtgg ccagtgaggt tgagctaaaa gacgcggtcg agcataacca 7860 acacccggtt gcaagaccgg ttgatggtgg tgttgtgctc ctgcgctccg cagttccttc 7920 gcttatagac gtcttaatct ccggcgctga tgcatctccc aagttactcg cccgccacgg 7980 gccgggaaac actgggatcg atggcacgct ttgggatttt gaggccgagg ccactaaaga 8040 ggaaattgca ctcagtgcgc aaataataca ggcttgtgac attaggcgcg gcgacgcacc 8100 tgaaattggt cttccttata agctgtaccc tgtcaggggc aaccctgagc gggtaaaagg 8160 agttttacag aatacaaggt ttggagacat accttataaa acccccagtg acactggaag 8220 cccagtgcac gcggctgcct gcctcacgcc caatgccact ccggtgactg atgggcgctc 8280 cgtcttggcc acgactatgc cctccggttt tgagttgtat gtaccgacca ttccagcgtc 8340 tgtccttgat tatcttgatt ctaggcctga ctgccccaaa cagttgacag agcacggctg 8400 tgaggacgcc gcattaagag acctctccaa gtatgacttg tccacccaag gctttgtttt 8460 acctggagtt cttcgccttg tgcgtaagta cctgtttgct catgtgggta agtgcccgcc 8520 cgttcatcgg ccttccactt accctgccaa gaattctatg gctggaataa atgggaacag 8580 gtttccacc aaggacatcc agagcgtccc tgaaatcgac gttctgtgcg cacaggccgt 8640 gcgggaaaac tggcaaactg ttaccccttg taccctcaag aaacagtatt gtgggaagaa 8700 gaagactagg acaatactcg gcaccaataa cttcattgcg ctggcccacc gggcagcgtt 8760 gagtggtgtc acccagggct tcatgaaaaa ggcgtttaac tcgcccattg ccctcggtaa 8820 aaacaaattt aaagagcttc agactccggt cttaggcagg tgccttgaag ctgatcttgc 8880 atcctgcgat cgctccacac ctgcaattgt ccgctggttt gccgccaatc ttctttatga 8940 acttgcctgt gctgaagagc acctgccgtc gtacgtgttg aactgctgcc acgacctact 9000 ggtcacgcag tccggcgcag taactaagag aggtggcctg tcgtctggcg acccgatcac 9060 ttctgtgtcc aacaccattt acagcttggt gatatatgca caacacatgg tgctcagtta 9120 ctttaaaagt ggtcaccctc atggccttct gtttctacaa gaccagctga agtttgagga 9180 catgctcaag gttcaacccc tgatcgtcta ttcggacgac ctcgtactgt atgccgagtc 9240 tcccaccatg ccaaactacc actggtgggt tgaacatctg aacctgatgc tgggttttca 9300 gacggaccca aagaagacag ccataacaga ctcgccatca tttctaggct gtaggataat 9360 aaatggacgc cagctcgtcc ctaaccgtga caggattctc gcggccctcg cctaccatat 9420 gaaggcaagc aatgtctctg aatactacgc ctcggcggct gcgatactca tggacagctg 9480 tgcttgttta gagtatgatc ccgaatggtt tgaagagctt gtagttggga tagcgcagtg 9540 tgcccgcaag gacggctaca gttttcccgg cccgccgttc ttcttgtcca tgtgggaaaa 9600 actcagatcc aatcatgagg ggaagaagtc cagaatgtgc gggtactgcg gggccccggc 9660 tccgtacgcc actgcctgtg gcctcgacgt ctgtatttac cacacccact tccaccagca 9720 ttgtccagtc atcatctggt gtggccaccc ggctggttct ggttcttgta gtgagtgcaa 9780 acccccccta gggaaaggca caagccctct agatgaggtg ttggaacaag tcccgtataa 9840 gcctccacgg actgtaatca tgcatgtgga gcagggtctc acccctcttg acccaggcag 9900 ataccagact cgccgcggat tagtctccgt taggcgtggc attagaggaa atgaggttga 9960 tctaccagac ggtgattatg ctagcaccgc cctactccct acttgtaaag agattaacat 10020 ggtcgctgtc gcctctaatg tgttgcgcag caggttcatc atcggcccgc ctggtgctgg 10080 gaaaacatac tggctccttc aacaggtcca ggatggtgat gtcatttaca cgccaactca 10140 ccagaccatg ctcgacatga ttagggcttt ggggacgtgc cggttcaacg tcccagcagg 10200 tacgacgctg caattccctg ccccctcccg taccggccct tgggttcgca tcctagccgg 10260 cggttggtgt cctggcaaga attccttcct ggatgaagca gcgtattgta atcaccttga 10320 tgtcttgagg cttcttagca aaactaccct cacctgtctg ggagatttca aacaactcca 10380 cccagtgggt tttgattctc attgctatgt ttttgacatc atgcctcaga ctcaactgaa 10440 gaccatctgg agatttggac agaatatctg tgatgccatt cagccagatt acagggacaa 10500 acttgtatcc atggtcaaca caacccgtgt aacctacgtg gaaaaacctg tcaagtatgg 10560 gcaagtcctc accccttacc acagggaccg agaggacggc gccatcacaa ttgactccag 10620 tcaaggcgcc acatttgatg tggttacact gcatttgccc actaaagatt cactcaacag 10680 gcaaagagcc cttgttgcta ttaccagggc aagacatgct atctttgtgt atgacccaca 10740 caggcaactg cagagcatgt ttgatcttcc tgcgaaaggc acacccgtca acctcgctgt 10800 gcaccgtgac gagcagctga tcgtgctaga tagaaataac aaagaatgca cggttgctca 10860 ggctctaggc aatggggata aattcagggc cacagacaag cgcgttgtag attctctccg 10920 cgccatttgt gcagatctgg aagggtcgag ctccccgctc cccaaggtcg cacacaactt 10980 gggattttat ttctcgcctg atttgacaca gtttgctaaa ctcccggtag aacttgcacc 11040 ccactggccc gtggtgacaa cccagaacaa tgaaaagtgg ccagaccggt tggttgctag 11100 ccttcgcccc gtccataagt atagccgcgc gtgcatcggt gccggctaca tggtgggccc 11160 ctcagtgttt ctgggcaccc ctggggttgt gtcatactat ctcacaaaat ttgtcagggg 11220 cgaggctcaa atgcttccgg agacagtctt cagcaccggc cgaattgagg tagattgccg 11280 tgagtatctt gatgaccggg agcgagaaat tgctgagtcc ctcccccatg ctttcattgg 11340 cgacgtcaaa ggcactaccg ttggaggatg tcaccatgtc acctccaaat accttccgcg 11400 cttccttccc aaggaatcag tcgcggtagt cggggtttca agccccggga aagccgcaaa 11460 agcagtttgc acattaacag atgtgtatct cccagatctc gaagcttacc tccacccaga 11520 gacccagtcc aagtgctgga aaatgatgtt ggacttcaag gaagttcgac tgatggtctg 11580 gaaggacaag acggcctatt ttcaacttga aggccgccat ttcacctggt accagcttgc 11640 aagctatgcc tcgtacatcc gagttcctgt taactctacg gtgtatttgg acccctgcat 11700 gggccctgcc ctttgcaaca gaagagttgt cgggtccact cattggggag ctgacctcgc 11760 agtcacccct tatgattacg gtgccaaaat catcctgtct agtgcatacc atggtgaaat 11820 gccccctggg tacaaaatcc tggcgtgcgc ggagttctcg cttgacgatc cagtgaggta 11880 caaacacacc tgggggtttg aatcggatac agcgtatctg tacgagttca ccggaaacgg 11940 tgaggactgg gaggattaca atgatgcgtt tcgtgcgcgc cagaaaggga aaatttataa 12000 ggccactgcc accagcatga ggtttcattt tcccccgggc cctgtcattg aaccaacttt 12060 aggcctgaat tgaaatgaaa tggggtccat gcaaagcctc tttgacaaaa ttggccaact 12120 ttttgtggat gctttcacgg aatttttggt gtccattgtt gatatcatta tatttttggc 12180 cattttgttt ggcttcacca tcgccggttg gctggtggtc ttttgcatca gattggtttg 12240 ctccgcgata ctccgtgcgc gctctaccat tcactctgag caattacaga agatcctatg 12300 aggccttcct ctcccagtgt caagtggaca tccccgcttg gggaaccaaa catcccctag 12360 ggatgctttg gcaccacaag gtatcgaccc tgatcgatga aatggtgtcg cgtcgaatgt 12420 accgcatcat ggaaaaggca ggacaggctg cctggaaaca ggtggtgacc gaggccacgc 12480 tgtctcggat tagtagcttg gatgtggtag ctcattttca gcaccttgcc gccattgaag 12540 ccgagacctg taaatattta gcatcccggc tgcccatgct acacaacctg cgcttgacag 12600 ggtcaaatgt aaccatagtg tataatagta ctttgaatca ggtgtttgct gtttttccaa 12660 cccccggttc ccggccaaag cttcatgatt ttcagcaatg gttaatagct gtacattcct 12720 ccatattttc ctctgttgca gcttcttgta ctctttttgt tgtgctgtgg ttgcggattc 12780 caatgctacg tactgctttt ggtttccgct ggttaggggc aatttttctt tcgaactcac 12840 agtgaattac acggtgtgcc cgccttgcct cacccgacag gcggccgcag agatctacga 12900 acccggtagg tctctttggt gtaggatagg gcacgatcga tgtggggagg atgaccacga 12960 cgagttaggg ttcatggtac cacctggcct ctccaacgaa ggtcatttgg ccagtgttta 13020 cgcctggttg gcgttcttgt cctttagcta cacagcccag ttccaccctg aaatatttgg 13080 gatagggaat gtgagcagag tttatgttga catcaagcat caattcatct gcgctgttca 13140 tgacggacag aacaccacct tgcctcgcca cgacaacatc tcagctgtgt tccagaccta 13200 ttaccaacat caggtcgacg gcggcaattg gtttcaccta gaatggttgc gtcccttctt 13260 ttcctcttgg ttggttttga acgtctcttg gtttctcagg cgttcgcctg caagccatgt 13320 ttcagttcga gtctttcaga cattaagacc aacaccaccg cggcggcagg ctttgctatc 13380 ctccaggaca tcagctgtct taggcatggc aactcaacct ctgaggcgtt tcgcaagatt 13440 cctcaatgcc gcacggcgat agggacacca gtgtatatca ccatcacagc caatgtaaca 13500 gatgagaatt atttacattc ttctgacctt ctcatgctct ctgcttgcct tttctatgct 13560 tctgagatga gcgaaaaggg atttaaggtg gtatttggca atgtgtcagg catcgtggct 13620 gtgtgtgtca attttaccag ctacgtccaa cacgtcaagg agtctaccca gcgctcctta 13680 gtggtcaatc atgtacggct gctccatttc atgacacctg agaccatgag gtgggcgact 13740 gttttagcct gtctttttgc cattctgttg gccatttgaa tgtttaagta tgttggggaa 13800 atgcttgacc gcgggctgtt gctcgcaatt gctttttttg tggtgtatcg tgccgtcttg 13860 ttttgttgcg atcgtcagcg ccaacgctag cagcagctca aatttacagc tgatttacaa 13920 cttgacgcta tgtgagctgg caggcacaga ttggctagct aacagatttg actgggcggt 13980 ggagtgtttt gttatttttc ctgtattgac tcacattgtc tcttatggtg ccctaaccac 14040 tagccacttc cttgacacag tcggtttggt cactgtgtct accgccggat ttgttcacgg 14100 gcggtatgtt ttgagtagca tttacgcggt ctgtgccctg gctgcgttga tttgcttcgt 14160 cattaggctt gcgaagaatt gcatgtcctg gcgctactca tgtaccagat ataccaactt 14220 tcttctagat accaagggca gactctaccg ttggcggtcg cctgtcatta tagagaaaag 14280 gggcaaagtt gaggtcgagg gtcaactaat cgaccccaaa agagttgtgc ttgatggttc 14340 cccggcaacc cctgtaacca gagtttcagc ggaacaatgg ggtcatcctt agatgacttc 14400 tgccatgata gcacagcccc acaaaaggtg ctcttggcgt tttctatcac ctacacgcca 14460 gtgatgatat acgccctaaa ggtaagtcgc ggccgactgc tagggctttt gcaccttttg 14520 atcttcctga attgtgcttt caccttcggg tatatgacat tcatgcactt tcagagtaca 14580 aataaggtcg cgctcactat gggagcagta gtggcactcc tttggggggt gtactcagcc 14640 atagaaacct ggagattcat cacctccaga tgccgtttgt gcttgctagg ccgcaagtac 14700 attctggccc ctgcccacca cgttgaaagt gccgcaggct ttcatccgat tgcggcaaat 14760 gataaccacg catttgtcgt ccggcgtccc ggctctacta cggtcaacgg cacattggtg 14820 cccgggttga aaagcctcgt gttgggtggc agaaaagctg ttaaacaggg agtggtaaac 14880 cttgttaaat atgccaaata acaacggcaa gcagcagaag agaaagaagg gggatggcca 14940 gccagtcaat cagctgtgcc aaatgctggg caaaattatc gcccagcaaa accagtccag 15000 aggcaaggga ccgggaaaga aaaataagaa gaaaaacccc gagaagcccc atttccctct 15060 tgcggctgaa gatgatgtca gacatcactt tacccctagt gagcggcaat tgtgtctgtc 15120 gtcaatccag accgccttta atcaaggcgc tgggacctgc accctatcag attcagggag 15180 gataagttac actgtggagt ttagtttgcc tacgcatcat actgtgcgcc tgatccgtgt 15240 cacagcatca ccttcagcat gatgagctgg cattcttgtg gcatcccagt atttgaattg 15300 gaataatgcg tggtgaatgg cactgattga cattgtgcct tcaagtcacc tattcaatta 15360 gggcgaccgt gtgggggcaa gatttaattg gcgagaacca cacggccgaa attaaaaaaa 15420 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaacgtt aattaacgat gcccttgaga 15480 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 15540 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt 15600 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc 15660 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc 15720 gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg 15780 ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc cggcggcatc 15840 gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag 15900 cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc gctgatcgtc 15960 acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc 16020 gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg 16080 acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga attggagcca 16140 atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg 16200 cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg tcctggccac 16260 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta 16320 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa 16380 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg 16440 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct 16500 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga 16560 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta 16620 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta 16680 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa 16740 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac 16800 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg 16860 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 16920 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 16980 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 17040 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt 17100 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 17160 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 17220 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 17280 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 17340 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 17400 gtgggagaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 17460 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 17520 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 17580 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 17640 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 17700 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 17760 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 17820 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 17880 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 17940 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 18000 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 18060 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 18120 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 18180 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 18240 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 18300 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 18360 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc 18420 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 18480 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 18540 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 18600 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 18660 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac 18720 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 18780 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 18840 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 18900 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 18960 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 19020 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 19080 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 19140 gcgtatcacg aggccctttc gtcttcaaga attctcatgt ttgacagctt atcatcgata 19200 agcttcggac cgtaatacga ctcactatag g 19231 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LMY forward primer <400> 3 gtggatgctt tcacggagtt c 21 <210> 4 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> LMY reverse primer <400> 4 cacacttaat taacgttttt tttttttttt tttttttttt tttttttttt ttttttttaa 60 tttcggccgt gtggttc 77 <210> 5 <211> 603 <212> DNA <213> Artificial Sequence <220> <223> PRRS K418 GP5 <400> 5 atgttgggga gatgcttgac cgcgggctgt tgctcgcaat tgcttttttt gtggtgtatc 60 gtgccatctt gttttgttgc gatcgtcagc gccaacaaca gcagcagctc aaatttacag 120 ctgatttaca acttgacgct atgtgagctg aatggcacag attggctagc taacagattt 180 gactgggcgg tggagtgttt tgttattttt cctgtattga ctcacattgt ctcttatggt 240 gccctaacca ctagccactt ccttgacaca gtcggtttgg tcactgtgtc taccgccgga 300 tttgttcacg ggcggtatgt tttgagtagc atttacgcgg tctgtgccct ggctgcgttg 360 atttgcttcg tcattaggct tgcgaagaat tgcatgtcct ggcgctactc atgtaccaga 420 tataccaact ttcttctaga taccaagggc agactctacc gttggcggtc gcctgtcatt 480 atagagaaaa ggggcaaagt tgaggtcgag ggtaaaaaaa tcgacctcaa aagagttgtg 540 cttgatggtt ccgcggcaac ccctgtaacc agagtttcgg cggagcaatg gggtcatcct 600 tag 603

Claims (17)

DNA of a porcine reproductive and respiratory syndrome virus (PRRSV) chimeric mutant having the nucleotide sequence shown in SEQ ID NO: 1. The virus reverse transcribed the DNA of the pig genital respiratory syndrome virus chimeric mutant of paragraph 1. DNA of the porcine reproductive and respiratory syndrome virus (PRRSV) chimeric mutant having the nucleotide sequence shown in SEQ ID NO: 2. The virus reverse transcribed the DNA of the pig genital respiratory syndrome virus chimeric mutant of paragraph 3. A cell comprising the DNA of the porcine reproductive-respiratory syndrome virus chimeric mutant of claim 1. A cell comprising the DNA of the porcine reproductive or respiratory syndrome virus chimeric mutant of claim 3. 7. The cell of claim 6, wherein said cell is deposited under accession number KCCM 11458P. 7. The method of claim 5 or 6, wherein said cells are selected from the group consisting of Escherichia lt; / RTI &gt; cells. 7. A porcine reproductive-respiratory syndrome virus vaccine comprising the DNA of claim 1 or 3, or the RNA of claim 2 or 4. 6. A porcine reproductive or respiratory syndrome virus vaccine comprising the DNA of claim 3 or the RNA of claim 4. 11. The vaccine of claim 10, wherein the vaccine further comprises at least one selected from the group consisting of carriers, diluents, excipients, and adjuvants. 11. The vaccine of claim 10, wherein the DNA or RNA is inactivated, a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine. A method for producing a porcine reproductive / respiratory syndrome virus chimeric mutant comprising the step of producing a porcine reproductive / respiratory syndrome virus using the cells of any one of claims 5 to 7. A method for producing a porcine reproductive / respiratory syndrome virus vaccine using the cells of any one of claims 5 to 7. A method for producing a porcine reproductive / respiratory syndrome virus vaccine using the DNA of claim 1 or 3, or the RNA of claim 2 or 4. A method for producing a porcine reproductive / respiratory syndrome virus chimeric mutant having the DNA sequence represented by SEQ ID NO: 1 by combining the ORF1 region of the PRRSV NVSL 97-9785 mutant and the ORF2 to ORF7 of the PRRSV LMY mutant. 1) combining the ORF1 region of the PRRSV NVSL 97-9785 mutant and the ORF2 to ORF7 of the PRRSV LMY mutant; And
2) A method for producing a porcine reproductive / respiratory syndrome virus chimeric mutant having the DNA sequence of SEQ ID NO: 2, which comprises mutating a gene encoding GP5 in the base sequence combined in step 1).

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