阅读说明：本技术 一种猪繁殖与呼吸综合征二价灭活疫苗及制备方法 (Porcine reproductive and respiratory syndrome bivalent inactivated vaccine and preparation method thereof ) 是由 李少丽 王家福 尹忠良 朱国坡 邵攀峰 胡晓凤 于 2021-05-27 设计创作，主要内容包括：本发明公开了一种猪繁殖与呼吸综合征二价灭活疫苗及制备方法。一种猪繁殖与呼吸综合征二价灭活疫苗,包括灭活后的以下两种病毒：株号为PRRSV-ZJ/H株的猪繁殖与呼吸综合征病毒,保藏号为CCTCC NO：V202130,株号为PRRSV-ZJ/NB株的猪繁殖与呼吸综合征病毒,保藏号为CCTCC NO：V202140。本发明制备的二价灭活疫苗能有效防控当前田间的两大主要流行PRRS病毒类型,在自然感染或用PRRS弱毒活疫苗免疫的商品猪场加强免疫一针PRRS灭活疫苗,可明显提高病毒特异性中和抗体,降低感染猪体内病毒载量,迅速有效控制PRRS疫情,还能明显提高猪的日增重,增加猪场经济效益。(The invention discloses a porcine reproductive and respiratory syndrome bivalent inactivated vaccine and a preparation method thereof. A bivalent inactivated vaccine for porcine reproductive and respiratory syndrome comprises the following two inactivated viruses: the strain number of the porcine reproductive and respiratory syndrome virus is PRRSV-ZJ/H strain, and the preservation number is CCTCC NO: v202130, porcine reproductive and respiratory syndrome virus with strain number PRRSV-ZJ/NB strain, preservation number CCTCC NO: v202140. The bivalent inactivated vaccine prepared by the invention can effectively prevent and control two main epidemic PRRS virus types in the field at present, and strengthens immunity of one PRRS inactivated vaccine in a commercial pig farm which is naturally infected or immunized by the PRRS attenuated live vaccine, so that the virus specificity neutralizing antibody can be obviously improved, the virus load in an infected pig body is reduced, the PRRS epidemic situation is quickly and effectively controlled, the daily gain of the pig can be obviously improved, and the economic benefit of the pig farm is increased.)

1. A Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome) bivalent inactivated vaccine comprises the following two inactivated viruses:

the strain number of the porcine reproductive and respiratory syndrome virus is PRRSV-ZJ/H strain, and the preservation number is CCTCC NO: the voltage of the V202130,

the strain number of the porcine reproductive and respiratory syndrome virus is PRRSV-ZJ/NB strain, and the preservation number is CCTCC NO: v202140.

2. The bivalent inactivated vaccine for porcine reproductive and respiratory syndrome according to claim 1, wherein the mixing volume ratio of the virus solutions of the two viruses of the PRRSV-ZJ/H strain and the PRRSV-ZJ/NB strain is 1: 1.

3. The bivalent inactivated vaccine for porcine reproductive and respiratory syndrome according to claim 2, wherein the titer of the virus solution is not less than 10^7.0TCID₅₀/ml。

4. The bivalent inactivated vaccine for porcine reproductive and respiratory syndrome according to claim 2, wherein the virus fluids of both viruses are inactivated with a BEI agent; the inactivated virus solution was emulsified with ISA 28VG adjuvant.

5. A preparation method of a Porcine Reproductive and Respiratory Syndrome (PRRSV) valence inactivated vaccine is characterized by comprising the following steps: preparing virus liquid from PRRSV-ZJ/H strain and PRRSV-ZJ/NB strain, inactivating the virus liquid, and emulsifying with adjuvant to obtain the inactivated vaccine.

6. The method of claim 5, wherein the virus solution has a titer of no less than 10^7.0TCID₅₀/ml。

7. The method according to claim 5, wherein the mixing volume ratio of ZJ/H strain to ZJ/NB strain is 1: 1.

8. The method of claim 5, wherein the inactivation was performed using a BEI agent.

9. The method of claim 5, wherein the adjuvant species is ISA 28 VG.

10. The method of claim 9, wherein the ratio of inactivated virus fluid to adjuvant is 3: 1.

Technical Field

The invention relates to the field of biological products, in particular to a porcine reproductive and respiratory syndrome bivalent inactivated vaccine and a preparation method thereof.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS), also known as Porcine reproductive and respiratory syndrome, is an acute, highly infectious viral infectious disease in swine herds characterized by reproductive disorders and respiratory symptoms. The disease can cause the abortion and premature birth of sows at the later stage of gestation, dead and weak piglets, and dyspnea, high mortality and slow growth of piglets and growing-finishing pigs, and the pigs at all ages can be infected. The autopsy pigs have interstitial pneumonia and lymph node enlargement, wherein the virus content in tonsils, lungs and lymph nodes is higher.

The PRRSV (Porcine reproductive and respiratory syndrome virus) is a positive-strand small RNA virus, belongs to the family of arteriviruses, is a virus with an envelope, and has a diameter of 50-65 nm. The virus has American type and European type 2 serotypes, and the American type is basically separated from China. PRRSV grows primarily in alveolar macrophages, blood monocytes and MA104 vero cells and derivatives thereof in pigs.

Highly pathogenic PRRSV (HP-PRRSV) has a deletion of 30 non-contiguous amino acids in the Nsp2 region. The sequence comparison analysis of Nsp2 gene of a new PRRSV variant named as NAD 30-like shows that discontinuous deletion of 131 amino acids exists on the Nsp2 gene.

The genome similarity between the NAD 30-like vaccine strain and the PRRSV vaccine strain which is popular at present is only 85.5-89.5%, so that the cross protection of the commercial vaccine to the NAD30 which is popular at present is weaker, and the infection of the NAD 30-like strain occurs in the pig farms which immunize the classical PRRSV vaccine and the HP-PRRSV vaccine, which indicates that the commercial vaccine can not generate enough immune protection to pigs to a certain extent.

Due to the characteristics of easy mutation and recombination of PRRSV, the vaccine has frequent virulent return phenomenon. Especially NAD30, is easy to recombine with vaccine virus and field wild virus, so that PRRSV in pig farm is diversified. People separate the PRRS vaccine virus and the wild virus recombinant strain from the field disease pig farm, and the worry about the safety of the PRRS attenuated vaccine gradually appears.

Under the background that swine diseases are more complicated due to normalization of African swine fever, PRRSV inactivated vaccines with good safety are used in a large amount in pig farms with more and more scales, and good production performance is achieved. The immunization scheme of the live vaccine and the inactivated vaccine for supplementing the deficiency can generate higher neutralizing antibody, reduce the morbidity of swinery, improve the production result and ensure the healthy and sustainable development of the live pig industry in China.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a bivalent inactivated vaccine for porcine reproductive and respiratory syndrome and a preparation method thereof.

A bivalent inactivated vaccine for Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome) comprises the following two inactivated viruses:

the strain number of the porcine reproductive and respiratory syndrome virus is PRRSV-ZJ/H strain, and the preservation number is CCTCC NO: the voltage of the V202130,

the strain number of the porcine reproductive and respiratory syndrome virus is PRRSV-ZJ/NB strain, and the preservation number is CCTCC NO: v202140.

Preferably, the mixing volume ratio of the virus liquid of the PRRSV-ZJ/H strain and the PRRSV-ZJ/NB strain is 1: 1.

More preferably, the titer of the virus solution of both strains of virus is not less than 10^7.0TCID₅₀/ml。

More preferably, the viral fluids of both viruses are inactivated using BEI reagents; the inactivated virus solution was emulsified with ISA 28VG adjuvant.

The invention also provides a preparation method of the porcine reproductive and respiratory syndrome bivalent inactivated vaccine, which comprises the following steps: preparing virus liquid from PRRSV-ZJ/H strain and PRRSV-ZJ/NB strain, inactivating the virus liquid, and emulsifying with adjuvant to obtain the inactivated vaccine. Preferably, the titer of the virus solution of the two viruses is not less than 10^7.0TCID₅₀And/ml. Preferably, the mixing volume ratio of ZJ/H strain to ZJ/NB strain is 1: 1. Preferably, the inactivation is performed using a BEI agent. Preferably, the adjuvant class is ISA 28 VG. More preferably, the mass ratio of the inactivated mixed virus solution to the adjuvant is 3: 1.

Compared with the prior art, the invention has the beneficial effects that:

the characteristic of strong tropism of PRRSV to PBMC cells is utilized, the PRRSV is separated by adopting the porcine Peripheral Blood Mononuclear Cells (PBMC), the pig is not required to be dissected, the operation is simple and convenient, and the method is a good choice for effectively separating certain viruses which are initially uncertain whether to adapt to subculture cells. After virus is separated from PBMC cells, Marc145 passage cells are used for carrying out adaptive culture on PRRSV isolates, and the vaccine virus liquid is conveniently prepared in a later period in an industrialized mode.

For the booster immunity of the PRRS inactivated vaccine, the immune protection of the homologous strain is stronger. The PRRS bivalent inactivated vaccine group prepared by the invention contains two types of viruses including ZJ/H strain and ZJ/NB strain, and can effectively prevent and control two major epidemic PRRS virus types in the field at present. Meanwhile, the immunization of the PRRS inactivated vaccine can reduce the phenomenon that the abuse of PRRS live vaccine leads to the more complex porcine reproductive and respiratory syndrome in pig farms, and is a necessary way for purifying the porcine reproductive and respiratory syndrome in original pig farms and pig farms. In addition, a PRRS inactivated vaccine is enhanced and immunized in a commercial pig farm naturally infected or immunized by a PRRS attenuated live vaccine, so that the virus specificity neutralizing antibody can be obviously improved, the virus load in an infected pig body is reduced, the PRRS epidemic situation is quickly and effectively controlled, the daily gain of the pig can be obviously improved, and the economic benefit of the pig farm is increased.

Drawings

FIG. 1 is a PBMC cytogram.

FIG. 2 is an electrophoretogram separating HP-PRRSV and NAD 30-like strains, wherein lane M: DL2000 Marker; lane 1: PRRSV CH-1a (positive control); lane 2: PRRSV ZJ/H strain; lane 3: PRRSV ZJ/NB strain; lane 4: and (5) negative control.

FIG. 3 is a graph showing the results of detection of neutralizing antibodies in different groups of test pigs before and after immunization and challenge.

FIG. 4 is a graph showing the results of the virus load changes in the serum of different groups of pigs tested at different time periods before and after challenge.

FIG. 5 is a graph showing the results of the daily gain change measurements on pigs in each survival test before and after challenge.

Detailed Description

Example 1: isolation and identification of PRRSV epidemic strains

(1) Viruses, cells and vaccines

PRRSV isolates epidemic strains ZJ/H and ZJ/NB, PBMC cells, Marc145 cells, a porcine peripheral blood mononuclear cell isolate kit, a highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain) and a porcine reproductive and respiratory syndrome inactivated vaccine (CH-1a strain).

And (5) collecting and processing pathological materials. Taking fresh lungs and lymph nodes of clinically suspected blue ear diseased pigs, adding cold PBS for grinding, centrifuging the grinding liquid for 10min at 3000r/min, taking supernatant, freezing and thawing for 2 times, and filtering by a 0.22 mu m bacteria filter to obtain suspected PRRSV stock solution.

Preparation of PBMC cells. Firstly, slowly extracting equal amount of anticoagulation (whole blood: anticoagulant is 6: 1) by using an anticoagulation tube added with anticoagulant, slightly inverting and uniformly mixing, taking a 15ml centrifuge tube, and carefully and slowly adding the separating medium 1 and the separating medium 2 in sequence according to the specification of a pig peripheral blood mononuclear cell separating medium kit to prepare a gradient interface. The cell suspension was pipetted onto the layering liquid surface, 500g and centrifuged for 20 min. Carefully pipette the second layer into another 15ml centrifuge tube, add 10ml of wash solution to the resulting centrifuge tube, and mix the cells. 250g, centrifugating for 10min, and discarding the supernatant. The resulting cells were resuspended with a pipette by adding 5ml of a washing solution, 250g, centrifuged for 10min, and washed repeatedly 2 times to obtain cell pellets.

And (5) culturing the PBMC cells. Using 1640 culture medium containing 10% fetal calf serum and adding 1.5-3 × 10⁶Resuspending the cells at a density of one/ml, and plating the cells onto the fine cellsIn cell culture plates, 5% CO at 37 ℃₂Culturing adherent cells in an incubator, removing floating cells after 24 hours, wherein the adherent cells are separated mononuclear cells (PBMC), and supplementing new growth liquid for culture. The cells were small and mostly star-shaped as seen by the microscope (see FIG. 1).

PBMC cells primary isolation of PRRSV. Inoculating the suspected PRRSV virus stock solution into the prepared PBMC cells according to the proportion of 10%, and inoculating 5% CO at 37 DEG C₂And (3) incubating for 1h in an incubator, adding 1640 maintenance liquid containing 2% fetal calf serum, continuing to culture for 48-96 h, observing and recording cytopathic effect every day, freezing and thawing the cytopathic liquid with obvious pathological changes for 2 times, and freezing and storing at-70 ℃.

(2) PCR detection and identification of PRRSV.

NSP2 gene primers were designed and synthesized with reference to PRRSV strain gene sequences (GenBank accession number: EU807840.1) for PCR amplification of isolate genomic sequences. The primers were synthesized by Shanghai Bioengineering Co., Ltd.

An upstream primer: 5 '-ATGTTGTGCTTCCTGGGGTT-3';

a downstream primer: 5 '-GACAAATCTAGAGGCTCGTC-3'.

Sucking 1 μ L of reverse transcription Primer Random Primer 1 μ L, RNA template, acting at 70 deg.C for 5min, and ice-cooling for 2 min; adding 4.0uL of 5 XM-MLV Buffer, 2.0 muL of 10mmol/L dNTP and 1.0 muL of HPRI RNase inhibitor, and acting at 37 ℃ for 5 min; centrifuging at 1000r/min for 10s, adding M-MLV reverse transcriptase 1.0ul, mixing, soaking at 42 deg.C for 90min, inactivating reverse transcriptase at 70 deg.C for 10min, ice-cooling for 2min, and storing at-20 deg.C.

And (3) PCR reaction system: 10 × PCR Buffer (Mg)²⁺Plus) 2. mu.L, 2.5mmol/L dNTP Mix 2.0. mu. L, TaqDNA polymerase 0.5. mu.L, upstream and downstream primers 0.5-1.5. mu.L each, template 5.0. mu.L, sterile double distilled water to make up to 20. mu.L PCR amplification program: and (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 40s, annealing at 46-55 ℃ for 45s, extension at 72 ℃ for 1.5min, 35 cycles in total, and extension at 72 ℃ for 10 min. The PCR product was detected by electrophoresis on 1% agarose gel. The results show that specific PRRSV bands appear at 767bp and 464bp (see figure 2), and PCR products identified as positive are sent to Shanghai Bioengineering Co., Ltd for sequencing.

The sequence alignment result of Mega6.0 software shows that compared with the classical PRRSV CH-1a, a highly pathogenic PRRSV and a NAD 30-like strain are separated from the detected sample and are respectively named as ZJ/H strain and ZJ/NB strain. The sequence of the NSP2 gene amplified by the highly pathogenic PRRSV ZJ/H strain is shown as SEQ ID No. 1; the sequence of the NSP2 gene amplified by the NAD 30-like strain ZJ/NB strain is shown as SEQ ID No. 2.

Inoculating the isolated PRRSV with 5% of the Marc145 monolayer cells at 37 deg.C and 5% CO₂Incubating for 1h in an incubator, adding DMEM containing 2% fetal calf serum for continuous culture, harvesting virus liquid when 80% cells are piled up and shed, freezing and thawing for 2 times, and freezing and storing at-70 ℃. Following the same procedure, 2 passages were again blindly inoculated. The isolated PRRSV, starting with the second generation of cultures on Marc145 cells, exhibited typical cytopathic effects indicating that the virus is suitable for propagation on Marc145 cells.

The separated ZJ/H strain and ZJ/NB strain PRRSV viruses are used for detecting half of the infection amount (TCID) of the viruses according to a Reed-Muench method₅₀). As a result, the virus titer can reach 10^7.0TCID₅₀More than ml.

And (3) carrying out exogenous detection on the ZJ/H strain and the ZJ/NB strain according to Chinese veterinary pharmacopoeia, and the strain is qualified in detection and can be used as a candidate virus seed of the PRRSV inactivated vaccine for researching the immune efficacy.

The isolated ZJ/H strain and ZJ/NB strain PRRSV viruses are preserved.

The ZJ/H strain PRRSV virus is classified and named as a porcine reproductive and respiratory syndrome virus, the strain number is PRRSV-ZJ/H strain, the PRRSV-ZJ/H strain is preserved in China center for type culture Collection in Wuhan, China, the preservation date is 2021 year, 5 month and 8 days, and the preservation number is CCTCC NO: and V202130.

The ZJ/NB strain PRRSV virus is classified and named as porcine reproductive and respiratory syndrome virus, the strain number is PRRSV-ZJ/NB strain, the PRRSV-ZJ/NB strain is preserved in China center for type culture Collection in Wuhan, China, the preservation date is 2021 year, 5 month and 8 days, the preservation number is CCTCC NO: v202140.

Example 2: preparation of bivalent inactivated vaccine for porcine reproductive and respiratory syndrome

Preparation of virus liquid for vaccine. Taking the growth as a good monolayerDiscarding the old culture solution of Marc145 cells, respectively inoculating ZJ/H strain and ZJ/NB strain of porcine reproductive and respiratory syndrome virus seeds according to the proportion of 5% (V/V), and inoculating 5% CO at 37 deg.C₂After adsorbing for 1h in the incubator, adding DMEM maintenance solution containing 2% fetal calf serum to continue culturing. And (4) harvesting virus liquid when 80% of cells are piled up and shed, freezing and thawing for 2 times, and storing at-70 ℃.

Half the infectious amount (TCID) of the virus₅₀) The measurement of (1). Diluting the prepared virus solution with serum-free culture medium by 10 times, and collecting 10 times^-4、10^-5、10^-63 dilutions, respectively inoculated in 96-well Marc145 cells, 6 wells per dilution, 0.1 ml/well, 5% CO at 37 deg.C₂Observing the incubator for 5 days, and calculating TCID₅₀. The virus content of both viruses is not less than 10^7.0TCID₅₀/ml。

Inactivation of virus fluid for vaccines. Respectively adding the qualified virus liquid into BEI (Bei inactivation) with the final concentration of 5mmol/L, stirring and inactivating for 24h at 37 ℃, finally adding 5mmol/L sodium thiosulfate for neutralizing for 1h, and storing at 2-8 ℃.

Mixing the two inactivated virus antigens according to the volume ratio of 1: 1, slowly adding ISA 28VG adjuvant of Sebidae company according to the ratio of virus water phase antigen to adjuvant (mass ratio) of 3: 1, stirring at 1000rpm for 20min, and making into emulsifier. The prepared vaccine is qualified by physical property inspection.

Safety test of bivalent inactivated vaccine for porcine reproductive and respiratory syndrome:

5 healthy replacement gilts of 5 months old (neutralizing antibody titer is less than 1: 4) are used, 4ml of vaccine is injected into each head and neck muscle, and 14 days are continuously observed without adverse reaction. The vaccine has good safety.

Immune efficacy test of bivalent inactivated vaccine for porcine reproductive and respiratory syndrome:

the PRRSV antigen and antibody negative healthy susceptible replacement gilts are applied to carry out an immune efficacy test on the porcine reproductive and respiratory syndrome bivalent inactivated vaccine.

Protocol for experimental animals:

40 healthy replacement gilts (neutralizing antibody titer < 1: 4) of 5 months old are divided into AH total 8 groups. Groups A to F are immunized with commercial highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain) 2 ml/head. After 2 weeks, C, D groups immunized with the prepared porcine reproductive and respiratory syndrome bivalent inactivated vaccine (ZJ/H strain + ZJ/NB strain) 2 ml/head, E, F groups immunized with the commercial porcine reproductive and respiratory syndrome inactivated vaccine (CH-1a strain) 2 ml/head; A. b, G and group H immunophosphorus acid (PBS) 2 ml. After a second 14d immunization, all test pigs were grouped according to the following protocol and challenged with ZJ/H or ZJ/NB strains (10)^5.0 TCID₅₀) 1ml of the medicine is dripped into the nose per head, 2ml of the medicine is injected into the neck muscle, the continuous observation is carried out for 14 days, and all the live pigs are euthanized. The immune efficacy of the prepared porcine reproductive and respiratory syndrome bivalent inactivated vaccine on replacement gilts is evaluated according to clinical symptoms, the level of neutralizing antibodies, the serum viral load, the weight change before and after challenge and the like (Table 1).

Table 1 test protocol groupings

Clinical symptoms after immunity and toxic material attack. The A-F groups are normal in spirit and appetite after vaccine immunization, and have no adverse reactions such as body temperature rise and the like.

A, B, C, D, E and F pigs survived after being attacked by toxin, and B, E and F pigs suffered from diseases respectively, so that the body temperature of the pigs suffered from diseases is increased, the spirit and appetite of the pigs are reduced, and respiratory symptoms such as cough, asthma and the like are caused. The pigs of the two non-immune toxin-attacking control groups, G and H, all suffered from the disease, and had 2 deaths and 1 death respectively. The results show that although the PRRS live vaccine can generate better immune protection on ZJ/H, the attack protection efficiency on ZJ/NB is reduced, and if the enhanced immunity of the PRRS bivalent inactivated vaccine is carried out after the PRRS live vaccine is immunized, no macroscopic lesion appears after the attack, and the PRRS live vaccine provides good attack protection on pigs (Table 2).

TABLE 2 post-challenge clinical symptoms and gross anatomic pathologic changes

Note: the numerator is the number of the test pigs with diseases or survival, and the denominator is the total number of the test pigs.

The neutralizing antibody is an important reference index for evaluating the vaccine-induced humoral immune protection. The detection result shows that the neutralizing antibodies of all groups after 21d are negative (< 1: 4), compared with the neutralizing antibodies of 28d after the first immunization of the C, D, E and F four inactivated vaccines in the boosting group, the neutralizing antibodies of 7d after the challenge are obviously increased, wherein the highest neutralizing antibodies of the group C have the titer of 4.9log 2. After challenge, the neutralizing antibody level of the 14d inactivated vaccine booster group is slightly reduced, but still obviously higher than that of other test groups, and the neutralizing antibody titer of the F group vaccine strain induced serum is lower than that of other three groups of inactivated vaccine booster groups (see figure 3).

The test groups respectively take blood after 28d (before challenge), 7d and 14d after challenge after first immunity, and detect PRRSV virus load in serum, as a result, C, D and E groups do not detect PRRSV in the whole challenge period, and the virus load in serum after challenge is lower, about 10, after a, B and F groups are challenged^1.3～10^2.2copies/. mu.l; the virus load of G and H groups is higher and reaches 10^3.4copies/. mu.l or more. Compared with the PRRS live vaccine which is singly immunized, the PRRS inactivated vaccine has the advantages that the strengthened immunity, especially the PRRS bivalent inactivated vaccine with higher immune homology can accelerate the toxin expelling in the test pig body, shorten the toxin expelling period and effectively reduce the virus amount of the virus in the environment after natural infection (see figure 4).

Survival test pigs were weighed 28 days after initial immunization (before challenge) and 7 and 14 days after challenge, and the average daily gain was calculated. Average daily gain (weight on day after challenge-weight on day of challenge)/day after challenge/day of challenge. The daily gain weight of all experimental pigs 7d after challenge is reduced compared with that of the experimental pigs 28d after first immunization on the challenge day, wherein the daily gain of C, D, E enhanced immunization groups is not obviously reduced (less than 15G), while the average of A, B two live vaccine immunization challenge groups is reduced by about 167G, and the weights of G and H groups are increased negatively. The daily gain of A, B test pigs gradually recovered to normal after 14 days of challenge, C, D, E and F groups basically recovered to normal growth level, and the live test pigs of G and H groups still did not achieve the gain. It is shown that the boost of inactivated vaccine provides a guarantee for increasing the daily gain of pigs and shortening the marketing days (see fig. 5).

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