CN109897832B

CN109897832B - Porcine circovirus type 3 virus strain and application thereof

Info

Publication number: CN109897832B
Application number: CN201910248151.1A
Authority: CN
Inventors: 张文亮; 栗利芳; 刘新月; 李鹏宇; 肖进; 齐鹏; 宋芳; 李玲; 杨祎
Original assignee: China Animal Husbandry Industry Co Ltd
Current assignee: China Animal Husbandry Industry Co Ltd
Priority date: 2019-03-29
Filing date: 2019-03-29
Publication date: 2021-07-06
Anticipated expiration: 2039-03-29
Also published as: CN109897832A

Abstract

The invention discloses a porcine circovirus type 3 virus strain and application thereof. The porcine circovirus strain is named as porcine circovirus type 3 ZM-PCV3-14, and the preservation number is CGMCC No. 17290. The strain is separated from a swinery which newly breaks out in China and has the epidemic disease, represents the epidemic dominant strain in China at present, has good strain background, can be used as a strain for producing inactivated vaccines and a virus seed for inspection, provides materials for subsequent related experimental research, and lays a material foundation.

Description

Porcine circovirus type 3 virus strain and application thereof

Technical Field

The invention relates to the field of pathogenic microorganisms, belongs to the field of biological products for livestock, and relates to separation and identification of porcine circovirus type 3 virus and application of the porcine circovirus type 3 virus in inactivated vaccines.

Background

In 6 months 2015, a pig farm in northern carrelaine developed Porcine reproductive disorders, Porcine Dermatitis Nephrotic Syndrome (PDNS), which was shown to be caused by a new viral infection, namely Porcine circovirus type 3-PCV 3, Porcine circovirus type 3 (PCV 3). Further studies have shown that PCV3 is widely distributed throughout large farms in the United states, Korea, Italy, Poland, etc.

Porcine circovirus type 3 (PCV 3) belongs to the family of circoviridae, the genus of circovirus, and is in the form of a regular dodecahedron without a capsular sac. According to gene analysis of PCV3, the total length of PCV3 genome is 2000bp, the genome structure is similar to PCV1 and PCV2, the PCV3 genome mainly comprises three open reading frames of ORF1, ORF2 and ORF3, and ORF1 mainly encodes Rep protein; ORF2 mainly encodes Cap protein, PCV3Cap gene encodes 214 amino acid residues, PCV2Cap gene encodes 194 or 195 amino acid residues, and PCV3 has 19-20 amino acids less than Cap gene of PCV 2. According to antigenic analysis and structural prediction, the amino acid homology between PCV2 and PCV3Cap protein is lower than 40%, so PCV2 and PCV3 are presumed not to have immune cross-protection property. Circovirus has genetic diversity, infects a wide range of hosts, and can spread across populations. The research shows that the evolution relationship between PCV3 and canine circovirus is the most close, but the research reports that the evolution relationship between PCV3 and bat circovirus is relatively close.

PCV3 was detected in pigs in 2016 successively in China, 222 samples collected from 35 pig farms in 11 provinces and cities in China were detected by the university of agriculture in China, and the positive rate of PCV3 infection in the pig farms is up to 68.6%, which indicates that PCV3 is commonly existed in the pig farms in China and is mixed with other pathogens to infect the pigs. Meanwhile, it is also reported that PCV3 infection may cause vaccine immunization failure of PCV2, so detection and vaccine research aiming at PCV3 are necessary for prevention and control work of PCV 3.

Disclosure of Invention

One of the purposes of the invention is to provide a new isolated porcine circovirus type 3 (PCV 3) virus epidemic strain.

The invention also aims to provide an effective and safe Porcine circovirus type 3 (PCV 3) vaccine for pigs and a preparation method of the inactivated vaccine aiming at the defects of the existing Porcine circovirus biological products;

the preparation method adopted by the inactivated vaccine is simple and safe, and the vaccine has a good immune effect.

The above object of the present invention is achieved by the following technical solutions:

the invention firstly provides a Porcine circovirus type 3 (PCV 3) virus which is separated from a pig and is named as Porcine circovirus type 3 (PCV 893) ZM-PCV3-14CGMCC No. 17290. The international committee for virus classification classifies it as: the Porcine Circovirus (PCV) belongs to the genus circovirus of the circovirus family, and the ZM-PCV3-14CGMCC No.17290 strain of the Porcine circovirus (PCV 3) is isolated from a pig farm in Guangdong province where Porcine circovirus type 3 virus infection occurs. Has been preserved in China general microbiological culture Collection center (China academy of sciences, institute of microbiology, No. 3, West Lu No.1, Beijing, Chaoyang, and North Cheng) in 2019, 28.1.9 days, and the preservation number is CGMCC No. 17290.

The method for separating and culturing the porcine circovirus type 3 comprises the following specific steps:

collecting porcine circovirus type 3 infected pig lymph node sample, preparing into 1:5 suspension with sterile PBS containing 100U/ml penicillin and 100ug/ml streptomycin after shearing, grinding, vortex shaking and mixing uniformly, repeatedly freezing and thawing for 3 times, centrifuging for 10min at 10000g, filtering the supernatant with a 0.22 μm filter for sterilization, and subpackaging and freezing at-70 ℃ for later use;

will be 1 × 10⁶Inoculating the PK-15 cells to a six-hole cell culture plate, and washing twice by using PBS when the fusion degree of the PK-15 cells reaches 80%; inoculating PK-15 cells with 1ml of filtered porcine circovirus type 3 treatment solution, setting negative control holes, and culturing at 37 deg.C and 5% CO₂Adsorbing in the incubator for 2h, discarding the inoculum, adding 2ml DMEM maintaining solution containing 2% fetal calf serum, culturing at 37 deg.C for 24h, treating with 300mM D-glucosamine hydrochloride, culturing at 37 deg.C for 48h, continuously passaging for 3 times, transferring to 3 rd generation, and performing indirect immunofluorescence assay (IFA) verification.

The indirect immunofluorescence assay (IFA) verification method comprises the following steps:

the virus isolation solution was inoculated into PK15 cell monolayer in 96-well cell plate at 2 wells/sample at 200. mu.l/well, while setting blank cell control at 5% CO₂Culturing at 37 deg.C for 24 hr, treating with 300mM D-glucosamine, and culturing for 48 hr; the maintenance solution was discarded and PBS buffer (0.0)1M pH7.0) washing the cells 3 times; adding cold acetone (80% acetone, 20% double distilled water), 150 μ l/hole, and fixing at 4 deg.C for 30 min; removing acetone, drying at room temperature, and washing with PBS buffer solution for 1 time; adding PCV3 positive serum diluted at the ratio of 1:200, 50 mu l/hole, and incubating for 1h at the temperature of 37 ℃; remove the well fluid, use PBS buffer solution to wash 5 times; adding FITC-labeled rabbit anti-pig secondary antibody diluted at a ratio of 1:200, incubating at 37 ℃ for 1h at a concentration of 50. mu.l/well; remove the well fluid, use PBS buffer solution to wash 5 times; when observed under a fluorescence microscope, the cell control wells should show no fluorescence, and the cytoplasm of the virus-inoculated cell wells should be stained with a large amount of green fluorescent substance. See fig. 1. In FIG. 1, the positive results are the results of isolated porcine circovirus.

The invention also provides a preparation method of the vaccine of the virus, which comprises the following steps:

1) and (3) virus culture: inoculating the porcine circovirus ZM-PCV3-14CGMCC No.17290 to PK-15 cells for culture, and harvesting a virus culture;

2) inactivating the virus culture, repeatedly freezing and thawing the virus culture for three times, centrifuging, collecting supernatant, adding an inactivating agent, and inactivating the virus to obtain an inactivated virus solution;

3) mixing the inactivated porcine circovirus type 3 virus solution and an adjuvant according to a proportion, emulsifying, and preparing the porcine circovirus type 3 inactivated vaccine.

The preparation method of the vaccine composition comprises the following specific steps of step 1): using low sugar DMEM medium containing 10% fetal calf serum, 100U/ml penicillin, 100ug/ml streptomycin, 37 deg.C, 5% CO₂Culturing PK-15 cells in an adherent manner by using an incubator; when the fusion degree reaches about 80%, the culture solution is discarded, the porcine circovirus type 3 ZM-PCV3-14CGMCC No.17290 strain is inoculated, and then low-sugar DMEM culture medium containing 2% fetal calf serum, 100U/ml penicillin and 100g/ml streptomycin is used for culturing at 37 ℃ with 5% CO₂Culturing for 24h, treating with 300mM D-glucosamine hydrochloride, culturing at 37 deg.C for 48h, and collecting virus culture;

the inactivating agent in the step 2) is diethylene imine, the final concentration of the added diethylene imine in the virus liquid is 1mmol/L, and the inactivating condition is that the inactivation is carried out for 28 hours at 30 ℃; after inactivation, sodium thiosulfate solution was added to neutralize the divinyl imine, the final concentration of the added sodium thiosulfate solution being 0.02 g/ml. The inactivation effect is inoculated to PK-15 blind passage 3, and no lesion appears, so that the inactivation is qualified.

The adjuvant in the step 3) is ISA206 or ISA201, and the volume ratio of the inactivated virus liquid to the adjuvant is 1: 1.

The invention has the following positive beneficial effects:

the separated 3-type ZM-PCV3-14CGMCC No.17290 strain of the porcine circovirus is separated from a swinery which has newly developed the porcine circovirus in China and has the epidemic disease, represents the epidemic dominant strain in China at present, has good strain background, can be used as an inactivated vaccine for producing the strain and a virus seed for inspection, provides materials for subsequent related experimental researches, and lays a material foundation.

The vaccine composition has good immunogenicity, can induce animals to generate stronger neutralizing antibodies after immunization, and has higher protection rate on the virus attack of corresponding strains. As no biological products related to the vaccine of the pathogen exist in the market at home and abroad, the vaccine composition has strong product competitive advantage, can effectively prevent the spread and dissemination of PCV3 in swinery, reduces the economic loss caused by the disease, and has wide application prospect.

The preparation method adopted by the vaccine is quick, simple and safe.

Drawings

FIG. 1 shows immunofluorescence pictures (positive and negative) of porcine circovirus IFA.

FIG. 2 is a PCR plot of the DNA of porcine circovirus type 3 ZM-PCV3-14CGMCC No.17290 in a tissue disease; lane 1 is the DNA of porcine circovirus type 3 ZM-PCV3-14CGMCC No.17290, lane 2 is the negative result, and lane M is the molecular weight marker.

FIG. 3 shows a specific target fragment of porcine circovirus type 3 ZM-PCV3-14CGMCC No. 17290. Lane 1 is a specific fragment of porcine circovirus type 3 ZM-PCV3-14CGMCC No.17290,

lanes

2 and 3 are negative results, and lane M is molecular weight marker.

FIG. 4 shows that the ZM-PCV3-14CGMCC No.17290 infection of porcine circovirus type 3 causes obvious cell vacuolation.

Detailed Description

Example 1: separation and identification of porcine circovirus type 3 ZM-PCV3-14CGMCC No. 17290.

1.1 Experimental materials

The disease material sample used in the experiment is from a pig farm with porcine circovirus in Guangdong province, and the collection time is 1 month in 2018; porcine Kidney cell line (Porcine Kidney, PK-15) was purchased from American type culture center ATCC.

1.2 primer design

2 pairs of specific primers were designed using Primier 5 with reference to PCV3KY965242.1 gene series published in GenBank, and the primers were synthesized by Biotechnology engineering (Shanghai) GmbH. The nucleotide sequence of the specific primer is as follows:

PCV3-F-1:5′-TAGTATTACCCGGCACCTC-3′；

PCV3-R-1:5′-GCCCGGCACCAAAATGAGAC-3′；

PCV3-F-2：5′-TTAGAGAACGGACTTGTAAC-3′；

PCV3-R-2：5′-ACAGCTGGCACATACTACAC-3′；

1.3 Collection and treatment of pathological Material

Collecting a lymph node sample with porcine circovirus disease, preparing a suspension of 1:5 by using sterile PBS containing 100U/ml penicillin and 100g/ml streptomycin after shearing, grinding the suspension by using a mortar to homogenate, carrying out vortex oscillation and uniform mixing, repeatedly freezing and thawing for 3 times, centrifuging for 10min at 10000 Xg, taking a supernatant, filtering and sterilizing the supernatant by using a 0.22 mu m filter, and subpackaging the filtrate at-70 ℃ for later use;

1.4 DNA detection of PCV in tissue specimens

DNA was extracted using a commercial DNA extraction kit and run according to the product instructions.

The PCR amplification reaction system is 25 mu l, wherein the PCR amplification reaction system contains upstream primer PCV 3-F-11 mu l, downstream primer PCV 3-R-11 mu l, PCR enzyme MIX 12.5 mu l, sterile distilled water 9.5 mu l and template DNA1 mu l. Circulating reaction parameters: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45 seconds, annealing at 54 ℃ for 30 seconds, and extension at 72 ℃ for 2 minutes for 34 cycles; final extension at 72 ℃ for 10 min.

The PCR products were detected by electrophoresis on a 1% agarose gel (see FIG. 2 for results). As can be seen from FIG. 2, a band of about 2000bp was amplified, corresponding to the expected size of the amplified fragment.

And recovering the target fragment of the PCR product by using a glue recovery and purification kit, cloning the target fragment to a pMD-18T vector, and sequencing the target fragment by using a biological engineering (Shanghai) corporation. The sequence is shown as a sequence 1 in a sequence table, the sequence result is subjected to manual proofreading and sequence splicing, and similarity comparison is carried out in an NCBI database by using a Blast program, so that the sequence gene has the highest homology with the known sequence of PCV3 and has 98-99% similarity, and the result proves that the nucleic acid of PCV3 in the disease is positive.

1.5 isolation and culture of the Virus

Will be 1 × 10⁶Inoculating the PK-15 cells to a six-hole cell culture plate, and washing twice by using PBS when the fusion degree of the PK-15 cells reaches 80%; inoculating PK-15 cells with the porcine circovirus type 3 virus filtrate frozen for later use in the step 1.3, wherein the inoculation amount is 1ml, meanwhile, a negative control hole is arranged, adsorbing the cells in a 5% CO2 incubator at 37 ℃ for 2h, discarding the inoculum, adding 2ml of DMEM maintenance solution containing 2% fetal bovine serum, culturing the cells at 37 ℃ for 24h, treating the cells with 300mM D-glucosamine hydrochloride, culturing the cells at 37 ℃ for 48h, continuously carrying out passage 3 times, and carrying out indirect immunofluorescence assay (IFA) verification after the cells are transferred to the 3 rd generation. PCV3 inoculated cell wells were stained with a large amount of green fluorescent substance, and negative control cells should show no fluorescence.

the virus isolation solution was inoculated into PK15 cell monolayer in 96-well cell plate at 2 wells/sample at 200. mu.l/well, while setting blank cell control at 5% CO₂Culturing at 37 deg.C for 24 hr, treating with 300mM D-glucosamine, and culturing for 48 hr; the maintenance solution was discarded, and the cells were washed 3 times with PBS buffer (0.01M pH7.0); adding cold acetone (80% acetone, 20% double distilled water), 150 μ l/hole, and fixing at 4 deg.C for 30 min; removing acetone, drying at room temperature, and washing with PBS buffer solution for 1 time; adding PCV3 positive serum diluted at the ratio of 1:200, 50 mu l/hole, and incubating for 1h at the temperature of 37 ℃; remove the well fluid, use PBS buffer solution to wash 5 times; adding FITC-labeled rabbit anti-pig secondary antibody diluted at a ratio of 1:200, incubating at 37 ℃ for 1h at a concentration of 50. mu.l/well; remove the well fluid, use PBS buffer solution to wash 5 times; in thatWhen observed under a fluorescence microscope, the cell control hole has no fluorescence, and the cytoplasm of the virus-inoculated cell hole has a large amount of green fluorescent substance staining. See fig. 1. In FIG. 1, the positive results are the results of isolated porcine circovirus.

Meanwhile, PCR amplification is carried out on the cell culture by using porcine circovirus type 3 virus specific primers PCV3-F-2 and PCV3-R-2, a PCV3 inoculated cell can detect an obvious PCV3 specific target fragment (shown in figure 3), and a corresponding nucleic acid fragment is not amplified in a non-inoculated PK-15 cell negative control group. The virus is shown to be adapted to PK-15 cells and can be stably propagated on the cells, and a PCV3 strain capable of being stably passaged is obtained through isolation.

The strain is named as porcine circovirus type 3 ZM-PCV3-14CGMCC No. 17290. The strain is preserved in China general microbiological culture Collection center (address: China academy of sciences, No. 3, West Lu No.1, Beijing, Chaoyang, and North Chen) in 2019, 1 month and 28 days, and the preservation number is CGMCC No. 17290.

1.6 animal regression test

10 21-28 days old PCV2, PCV3ELISA antibody, PCV2 and PCV3 antigen-negative weaned piglets are selected and randomly divided into 2 groups and 5 groups, wherein the first group uses porcine circovirus type 3 ZM-PCV3-14 strain (10-10 ZM-PCV3-14 strain)^6.0TCID₅₀/ml) was challenged with 1ml nasal drop per pig, 2ml intramuscular, and the second group was inoculated with cell culture by the same method. After toxin attack, clinical observation is carried out for 25 days, during which the piglets in the toxin attack group are all lack of food, cyanosis of the skin and continuous body temperature rise for 3-11 days; serum viral load tests showed that PCV3 viral load was significantly elevated and 1 died. After the pigs are subjected to autopsy 25 days, the pigs in the offending group have congestion, swelling and the like of a plurality of lymph nodes and spleens, congestion, fleshiness, edema and the like of the lung at different degrees, and yellow kidney and bleeding spots or necrotic foci and the like. The details are shown in Table 1.

TABLE 1 porcine circovirus type 3 ZM-PCV3-14 animal regression test results

Example 2: determination of the Virus Titers of the porcine circovirus type 3 Virus Strain ZM-PCV3-14

Inoculating the separated virus solution to PK15-B1 cell monolayer, adsorbing at 37 deg.C for 30 min, adding DMEM cell maintenance solution containing 2% calf serum, culturing at 37 deg.C for 60 hr, and continuously passaging to 20 th generation and storing at-20 deg.C. PCV3 was maintained in DMEM 10^-1～10^-8Serial dilutions were plated in 96-well plates of PK15-B1 cell monolayer in 4 wells at 100 μ l per dilution. Meanwhile, a negative control is set up, the mixture is put into a CO2 incubator and cultured for 12h at 37 ℃, treated with 300mM D-glucosamine hydrochloride and cultured for 48h at 37 ℃. Fixing cells with absolute ethanol, determining the number of wells containing green fluorescent substance cells at each dilution by IFA method, and calculating TCID of virus by Reed-Muench method₅₀. The results show that the virus valence of the ZM-PCV3-14 strain of the porcine circovirus type 3 virus is 10^6.0TCID₅₀/ml。

Example 3: preparation of porcine circovirus type 3 ZM-PCV3-14 strain inactivated vaccine

3.1 Virus culture

Using low sugar DMEM medium containing 10% fetal calf serum, 100U/ml penicillin, 100g/ml streptomycin, T225cm²Cell culture flask, 37 deg.C, 5% CO₂Culturing PK-15 cells in an adherent manner by using an incubator; when the fusion degree reaches about 80%, removing the culture solution, inoculating the porcine circovirus type 3 ZM-PCV3-14 strain, then culturing for 12h at 37 ℃ and 5% CO2 by using a low-sugar DMEM medium containing 2% fetal calf serum, 100U/ml penicillin and 100g/ml streptomycin, treating with 300mM D-glucosamine hydrochloride, and continuously culturing for 48h at 37 ℃ to obtain a virus culture; repeatedly freezing and thawing for 3 times, centrifuging at 10000 Xg for 10min, removing cell debris, collecting supernatant to obtain porcine circovirus type 3 virus liquid, and standing at-80 deg.C for use.

3.2 inactivation of viral fluid and inactivation test

In virus liquid with qualified virus content (preferably more than or equal to 10)^6.0TCID₅₀/ml) adding an inactivating agent of divinyl imine to ensure that the final concentration of the divinyl imine is 1mmol/L, placing the divinyl imine in a constant-temperature shaking table at 30 ℃ and at 80rpm, and inactivating for 28 hours; after inactivation, sodium thiosulfate solution was added to neutralize the divinyl imine, the final concentration of the added sodium thiosulfate solution being 0.02 g/ml. Will be 1 × 10⁶Inoculating the PK-15 cells to a six-hole cell culture plate, and washing twice by using PBS when the fusion degree of the PK-15 cells reaches 80%; diluting inactivated virus solution 10 times with maintenance solution, inoculating 500 μ l, setting negative control well, and incubating at 37 deg.C and 5% CO₂Adsorbing for 2h in the incubator, discarding the inoculum, adding 2ml of maintenance liquid, continuing to culture for 4-5 d, and observing the cell state every day. And (3) blind passage, extracting sample DNA from each cell culture, and detecting the porcine circovirus type 3 virus nucleic acid in the cells by PCR according to the step 1.4. If the results are negative, the virus liquid is completely inactivated. IFA detection is carried out according to the step 1.5, and the result shows that the product is positive if green fluorescence exists and negative if no green fluorescence exists. If the results are negative, the virus liquid is completely inactivated.

3.3 preparation of vaccine composition containing circovirus type 3 Virus ZM-PCV3-14 Strain

Mixing the completely inactivated ZM-PCV3-14 virus solution with ISA206 adjuvant according to the volume ratio of 1:1, and emulsifying the oil phase and the water phase under the conditions of 30 ℃ constant temperature shaking table, 120rpm and 15 min. And (3) after the mixing is complete, obtaining the vaccine composition, wherein the emulsification quality of the vaccine can reach the quality standard specified by the state.

Example 4: immunogenicity test of inactivated vaccine of ZM-PCV3-14 strain of circovirus type 3

4.1 circovirus type 3 ZM-PCV3-14 inactivated vaccine immunization

Antigen-antibody negative screening is carried out on piglets of 5-6 weeks old by the method of step 1.4 of example 1 and PCV3Cap protein (the gene sequence is sequence 2 in the sequence table, and the amino acid sequence is shown as sequence 3) expressed by coating escherichia coli, and 10 piglets with negative antigen-antibody are selected to be subjected to immunogenicity evaluation test of the inactivated vaccine of the circovirus type 3 virus ZM-PCV3-14 strain. Grouping condition: randomly selecting 5 piglets of the inactivated vaccine composition of the porcine circovirus type 3 ZM-PCV3-14 strain prepared in example 3, wherein each 1ml of the vaccine composition is selected; control group 5 heads were injected intramuscularly with PBS 1ml per head and neck. Measuring the body temperature every day from 0 to 7 days of immunization; continuously observing the appetite and mental state of the swinery for 14 days; and observing and touching whether the local part of the injection part has the tumor or not.

After the porcine circovirus type 3 ZM-PCV3-14 inactivated vaccine is used for immunizing piglets, no obvious body temperature rise phenomenon (table 2) occurs, and the pigs eat normally and have good mental state; the vaccine injection part has no pain and lumps after 5 days of immunization. The vaccine is proved to have good safety for the immunized piglets.

TABLE 2 animal body temperature monitoring watch after inactivated vaccine immunization

4.2 determination of inactivated vaccine-induced antibody of circovirus type 3 ZM-PCV3-14 strain

In order to evaluate the immune effect of the ZM-PCV3-14 strain inactivated vaccine, serum is collected at 14, 21, 28 and 35d after immunization, the porcine circovirus ELISA antibody and neutralizing antibody production state in the serum is evaluated by using PCV3Cap protein (the gene sequence is shown as sequence 2 in a sequence table, and the amino acid sequence is shown as sequence 3) expressed by coated escherichia coli and a neutralizing test, and the immunogenicity of the ZM-PCV3-14 strain inactivated vaccine is evaluated.

4.2.1 coating PCV3Cap protein expressed by Escherichia coli to carry out ELISA antibody detection, wherein the detection result shows that PCV3 antibody can be detected 14 days after vaccine immunization; then the antibody level gradually increases, 35 days after the first immunization, the arithmetic mean titer of the ELISA antibody of the immunization group reaches 1:3520, no antibody is detected in the control group, and the result is shown in Table 3.

TABLE 3 porcine circovirus type 3 ZM-PCV3-14 strain induces the dynamic change of antibody production of piglets

4.2.2 in order to further evaluate the titer of the antibody generated by the ZM-PCV3-14 strain inactivated vaccine induced piglets, the level of the antibody generated by the inactivated vaccine induced immunized piglets is detected by using a neutralization test technology. The method comprises the following specific steps:

1) the serum to be detected is heated at 56 ℃ for 30 minutes, centrifuged at 10000r/min for 5 minutes, the supernatant is carefully sucked out, and diluted by 2 times after being diluted by 10 percent DMEM medium at a ratio of 1: 4.

2) Respectively equivalent to 1000TCID₅₀ZM-PCV3-14 strain virus liquid is mixed, incubated at 37 ℃ for 1 hour, inoculated into a 96-well plate containing PK15-B1 cell monolayer, 100. mu.l/well, 4 wells are inoculated in parallel at each dilution, and a cell control well (10% DMEM medium added with 100. mu.l/well) and a virus control well (1000 TCID added with 100. mu.l/well) are simultaneously arranged₅₀PCV3 virus fluid).

3)37℃，5％CO₂The culture was carried out in the incubator (2) for 24 hours, treated with a MEM-maintaining solution containing 2mmol/L D-glucosamine hydrochloride, and further cultured for 24 hours.

4) Cells were fixed in absolute ethanol at 50. mu.l/well.

5) And (4) measuring the number of wells containing fluorescence in each dilution by using an indirect immunofluorescence method, wherein the cell control wells have no fluorescence, the virus control wells have fluorescence, and the test is established.

6) The maximum dilution of serum in the wells capable of reducing the number of specific fluorescent cells by 50% compared to the virus control wells was taken as the neutralization titer of the test serum, and the average value per group was calculated.

The data were collated after determination of the serum neutralizing antibody titres and the results are shown in table 4. As can be seen from the table 4, the ZM-PCV3-14 strain inactivated vaccine composition prepared in example 3 can induce the immunized piglet to generate specific antibodies after being immunized for 14d, and basically meets the immunization requirement; the 35d antibody continued to rise, with the highest antibody being able to reach 1: 35.2. While the control group did not detect neutralizing antibodies against porcine circovirus type 3 virus (table 4).

TABLE 4 porcine circovirus type 3 ZM-PCV3-14 strains induce the dynamic changes of neutralizing antibodies produced by piglets

Example 5: challenge protection test of inactivated vaccine of ZM-PCV3-14 strain of circovirus type 3

25 28-35 days old PCV2, PCV3ELISA antibody, PCV2 and PCV3 antigen-negative weaned piglets are selected and randomly divided into 5 groups, 5 groups/group, wherein the 1-3 groups are immune groups, wherein the 1 st group is immunized by injecting neck muscle to immunize 0.5ml of ZM-PCV3-14 strain inactivated vaccine composition prepared in the example 3, the 2 nd group and the 3 rd group are immunized by the same way respectively by 1ml and 2ml, the 4 th group is a challenge control group, is immunized by 1ml of PBS, and the 5 th group is a blank control group, and is not immunized and not challenged. 2 weeks after immunization, porcine circovirus type 3 ZM-PCV3-14 (10)^6.0TCID₅₀Per ml) and 2ml per pig by nasal drip and intramuscular injection. Blood was collected at 5, 15 and 25 days after challenge, serum was isolated and viremia was determined by Q-PCR. Slaughtering 25 days after challenge, and observing pathological changes.

5.1 Observation of clinical symptoms and pathological changes

The results are shown in Table 5. After the virus attack is observed for 25 days, no obvious clinical manifestation is seen in the three doses of inactivated vaccine immunized pigs in the vaccine group, only one excessive body temperature rise (more than or equal to 40 ℃) appears in individual pigs, and the pigs recover to be normal after the continuous 1-2 days; 10-14 days after the PCV3 of the challenge control group, the temperature of 4 pigs is raised by 40.2-40.8 ℃ for more than 3 days, wherein 3 pigs are disordered and slightly reduced in appetite, and are recovered to be normal after 3 days. The autopsy test shows that 4/5 pigs in the toxicity attacking control group have lymph node swelling or bleeding, 3/5 pigs have lung bleeding, 2/5 pigs have spleen bleeding, and the comprehensive clinical symptoms are judged to be 80% of diseases. The lymph node tissues after the immunization of the group 2 and the group 3 have no obvious abnormality, and are judged to be 100% protection by combining clinical symptoms; in

group

1, 1/5 pig lymph node tissues showed lymphadenectasis, and 80% protection was observed in the combined clinical symptoms. The lymph node tissues of the blank control group have no obvious abnormality, and the comprehensive clinical symptoms are judged to be no disease.

TABLE 5 clinical symptoms and pathological changes following immunochallenge with porcine circovirus type 3 ZM-PCV3-14 vaccine

5.2 post-challenge PCV3 viremia detection

Collecting serum on 5 th, 15 th and 25 th days after the challenge, extracting DNA, and detecting PCV3 by a PCR method, wherein the result is as follows: no virus was detected by blank control porcine PCR; the virus copy number is detected at different time after the challenge, and the vaccine immunization groups have no significant difference (P >0.05), but have significant difference (P <0.05) with the challenge control group. And 5-25 days after the virus attack, the virus copy number of the immune group is not obviously increased, and the virus copy number of the virus attack control group is obviously increased along with time. Indicating that the 3 groups of vaccines can reduce virus replication after immunization. The results are shown in Table 6.

TABLE 6 PCV3Real-time PCR assay results in serum at different times after challenge

Note: different letters (a, b, c, d, e, f) at the same time indicate significant differences between the different groups (P < 0.05).

The porcine circovirus type 3 virus vaccine composition provided by the invention is proved to be capable of well protecting animals from being attacked by the porcine circovirus type 3 virus, and the data lays a certain theoretical and practical foundation for the clinical use of the inactivated vaccine.

Sequence listing

<110> Zhongmu industries GmbH

<120> porcine circovirus type 3 virus strain and application thereof

<130> WHOI190019

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2000

<212> DNA

<213> Porcine circovirus (Portone virous)

<400> 1

tagtattacc cggcacctcg gaacccggat ccacggaggt ctgtagggag aaaaagtggt 60

atcccattat ggatgctccg caccgtgtga gtggatatac cgggcagtgg atgatgaagc 120

ggcctcgtgt tttgatgccg caggacgggg actggataac tgagtttttg tggtgctacg 180

agtgtcctga agataaggac ttttattgtc atcctatttt aggtccggag ggaaagcccg 240

aaacacaggt ggtgttttac gataaacaac tggaccccga ccgagtggga atctattgtg 300

gagtgtggag gcagtatagc gagatacctt attatcggca aagaggttgg aaaaagcggt 360

accccacact tgcaagggta cgtgaatttc aagaacaaaa ggcgactcag ctcggtgaag 420

cgcttacccg gatttggtcg ggcccatctg gagccggcga gggggagcca caaagaggcc 480

agcgagtatt gcaagaaaga gggggattac ctcgagattg gcgaagattc ctcttcgggt 540

accagatcgg atcttcaagc agcagctcgg attctgacgg agacgtcggg aaatctgact 600

gaagttgcgg agaagatgcc tgcagtattt atacgctatg ggcggggttt gcgtgatttt 660

tgcggggtga tggggttggg taaaccgcgt gattttaaaa ctgaagttta tgtttttatt 720

ggtcctccag gatgcgggaa aacccgggaa gcttgtgcgg atgcggctgc gcgggaattg 780

cagttgtatt tcaagccacg ggggccttgg tgggatggtt ataatgggga gggtgctgtt 840

attttggatg atttttatgg gtgggttcca tttgatgaat tgctgagaat tggggacagg 900

taccctctga gggttcctgt taagggtggg tttgttaatt ttgtggctaa ggtattatat 960

attactagta atgttgtacc ggaggagtgg tattcatcgg agaatattcg tggaaagttg 1020

gaggccttgt ttaggaggtt cactaaggtt gtttgttggg gggagggggg ggtaaagaaa 1080

gacatggaga cagtgtatcc aataaactat tgattttatt tgcacttgtg tacaattatt 1140

gcgttggggt gggggtattt attgggtggg tgggtgggca gccccctagc cacggcttgt 1200

cgcccccacc gaagcatgtg ggggatgggg tccccacatg cgagggcgtt tacctgtgcc 1260

cgcacccgaa gcgcagcggg agcgcgcgcg aggggacaca gcttgtcgcc accggagggg 1320

tcagatttat atttattttc acttagagaa cggacttgta acgaatccaa acttctttcg 1380

tgccgtagaa gtctgtcatt ccagtttttt ccgggacata aatgctccaa agcagtgccc 1440

cccattgaac ggtggggtca tatgtgttga gccatggggt gggtctggag aaaaagaaga 1500

ggctttgtcc tgggtgagcg ctggtagttc ccgccagaat tggtttgggg gtgaagtaac 1560

ggctgtgttt ttttttagaa gtcataactt tacgagtgga actttccgca taagggtcgt 1620

cttggagcca agtgtttgtg gtccaggcgc cgtctagatc tatggctgtg tgcccgaaca 1680

tagtttttgt ttgctgagct ggagaaatta cagggctgag tgtaactttc atctttagta 1740

tcttataata ttcaaagcta atcgcagttt cccactcgtt taggcgggta atgaagtggt 1800

tggcgtgcca gggcttgtta ttctgaggag ttccaacgga aatgacgttc atggtggagt 1860

atttctttgt gtagtatgtg ccagctgtgg gcctcctaat gaatagtctt cttctggcat 1920

agcgccttct gtggcgtcgt cgtctccttg ggcggggtct tcttctaaat atagctctgt 1980

gtctcatttt ggtgccgggc 2000

<210> 2

<211> 543

<212> DNA

<213> Porcine circovirus (Portone virous)

<400> 2

acagctggca catactacac aaagaaatac tccaccatga acgtcatttc cgttggaact 60

cctcagaata acaagccctg gcacgccaac cacttcatta cccgcctaaa cgagtgggaa 120

actgcgatta gctttgaata ttataagata ctaaagatga aagttacact cagccctgta 180

atttctccag ctcagcaaac aaaaactatg ttcgggcaca cagccataga tctagacggc 240

gcctggacca caaacacttg gctccaagac gacccttatg cggaaagttc cactcgtaaa 300

gttatgactt ctaaaaaaaa acacagccgt tacttcaccc ccaaaccaat tctggcggga 360

actaccagcg ctcacccagg acaaagcctc ttctttttct ccagacccac cccatggctc 420

aacacatatg accccaccgt tcaatggggg gcactgcttt ggagcattta tgtcccggaa 480

aaaactggaa tgacagactt ctacggcacg aaagaagttt ggattcgtta caagtccgtt 540

ctc 543

<210> 3

<211> 181

<212> PRT

<213> Porcine circovirus (Portone virous)

<400> 3

Thr Ala Gly Thr Tyr Tyr Thr Lys Lys Tyr Ser Thr Met Asn Val Ile

1 5 10 15

Ser Val Gly Thr Pro Gln Asn Asn Lys Pro Trp His Ala Asn His Phe

20 25 30

Ile Thr Arg Leu Asn Glu Trp Glu Thr Ala Ile Ser Phe Glu Tyr Tyr

35 40 45

Lys Ile Leu Lys Met Lys Val Thr Leu Ser Pro Val Ile Ser Pro Ala

50 55 60

Gln Gln Thr Lys Thr Met Phe Gly His Thr Ala Ile Asp Leu Asp Gly

65 70 75 80

Ala Trp Thr Thr Asn Thr Trp Leu Gln Asp Asp Pro Tyr Ala Glu Ser

85 90 95

Ser Thr Arg Lys Val Met Thr Ser Lys Lys Lys His Ser Arg Tyr Phe

100 105 110

Thr Pro Lys Pro Ile Leu Ala Gly Thr Thr Ser Ala His Pro Gly Gln

115 120 125

Ser Leu Phe Phe Phe Ser Arg Pro Thr Pro Trp Leu Asn Thr Tyr Asp

130 135 140

Pro Thr Val Gln Trp Gly Ala Leu Leu Trp Ser Ile Tyr Val Pro Glu

145 150 155 160

Lys Thr Gly Met Thr Asp Phe Tyr Gly Thr Lys Glu Val Trp Ile Arg

165 170 175

Tyr Lys Ser Val Leu

180

Claims

1. A strain of porcine circovirus type 3 virus is characterized in that: the strain is named as porcine circovirus type 3 virus ZM-PCV3-14, and the preservation number of the strain in the China general microbiological culture Collection center is CGMCC number 17290.

2. Use of the porcine circovirus type 3 virus of claim 1 for the preparation of a vaccine for the prevention and treatment of diseases caused by porcine circovirus type 3 virus.

3. Use according to claim 2, characterized in that: the vaccine is an inactivated vaccine.

4. The vaccine for preventing and treating diseases caused by porcine circovirus type 3 virus has the active component of inactivated porcine circovirus type 3 virus ZM-PCV3-14 with the preservation number of CGMCC number 17290.

5. The vaccine of claim 4, wherein: the vaccine comprises inactivated porcine circovirus type 3 virus ZM-PCV3-14 with the preservation number of CGMCC number 17290 and a pharmaceutically acceptable adjuvant.

6. A process for the preparation of a vaccine according to claim 4 or 5 for the prevention and treatment of diseases caused by porcine circovirus type 3, comprising the steps of:

1) and (3) virus culture: inoculating the porcine circovirus type 3 virus ZM-PCV3-14 with the preservation number of CGMCC number 17290 to PK-15 cells for culture, and harvesting a virus culture;

3) mixing the inactivated porcine circovirus type 3 virus ZM-PCV3-14 virus liquid with the preservation number of CGMCC number 17290 and an adjuvant in proportion, emulsifying, and preparing the porcine circovirus type 3 virus inactivated vaccine.

7. The preparation method according to claim 6, wherein the inactivating agent is diethylene imine, the final concentration of the added diethylene imine in the virus solution is 1mmol/L, and the inactivating condition is 30 ℃ for 28 h; after inactivation, sodium thiosulfate solution was added to neutralize the divinyl imine, the final concentration of the added sodium thiosulfate solution being 0.02 g/ml.

8. The method according to claim 7, wherein the adjuvant is ISA206 or ISA201, and the volume ratio of the inactivated virus solution to the adjuvant is 1: 1.

9. The application of the porcine circovirus type 3 virus ZM-PCV3-14 with the preservation number of CGMCC number 17290 in establishing an animal model of the porcine circovirus type.