CN113862232A

CN113862232A - Method for obtaining high-toxicity seneca virus vaccine seed virus and application thereof

Info

Publication number: CN113862232A
Application number: CN202111113379.3A
Authority: CN
Inventors: 齐志涛; 方建国; 陈九连; 高艳华; 路荣; 刘玉梅; 刘东霞; 宋庆庆; 赵丽霞; 陈坚; 王玉雯; 马立峰; 张燕红; 杜宇荣; 李雪峰; 刘燕军
Original assignee: Jinyubaoling Bio Pharmaceutical Co ltd
Current assignee: Jinyubaoling Bio Pharmaceutical Co ltd
Priority date: 2021-09-18
Filing date: 2021-09-18
Publication date: 2021-12-31

Abstract

The invention discloses a method for obtaining high-toxicity seneca virus vaccine seed virus and application thereof, belonging to the technical field of biological products for livestock. The method provided by the invention comprises the steps of selecting the diseased tissue with the most serious clinical symptom expression of the diseased pig infected by the original Securin virus, carrying out antigen separation on the diseased tissue, and obtaining the Securin virus vaccine seed virus with high toxicity through cell culture and continuous multiple cloning and purification. The method can effectively obtain the seneca virus vaccine seed virus with high titer and strong immunogenicity, and can be used for producing the seneca virus vaccine with particularly ideal immune effect.

Description

Method for obtaining high-toxicity seneca virus vaccine seed virus and application thereof

Technical Field

The invention belongs to the technical field of biological products for livestock, and particularly relates to a method for obtaining high-toxicity seneca virus vaccine seed virus and application thereof, in particular to application in preparation of the seneca virus vaccine.

Background

Seneca Virus A (SVA) belongs to the genus Seneca virus of the family Picornidae, and can cause vesicular lesions or death of infected pigs after the infection of the pigs, thereby becoming a virus of major concern in the pig industry.

At present, the prevention and the treatment of diseases caused by the virus are mainly based on vaccine prevention, the selection of vaccine seed viruses is the most important link in the development process of the vaccine, and the seed viruses with high toxicity (expressed as high titer and high pathogenicity (namely high immunogenicity)) directly determine the immune effect of the vaccine. However, the current seneca virus vaccine seed viruses used in the preparation of seneca virus vaccines often show low titers (typically at 10)^7.0TCID₅₀About 0.1ml) and/or weak immunogenicity (namely, poor pathogenicity to the pigs of the animals), so that the prepared Seneca virus vaccine can not achieve a particularly ideal immune effect. For example, patent document CN110923211A (hereinafter referred to as document 1) discloses an Selcarbovirus isolate (CH-HLJ CGMCC No.18851) having a viral titer of 10^7.0TCID₅₀0.1ml, the pathogenicity rate to the animal pig is 80%, the vaccine can be used for preparing the Seneca virus vaccine according to the vaccine preparation procedure, and the prepared vaccine has the serum neutralizing antibody titer reaching 1:1024 at the maximum 28 days after the first immunization under the condition of 3ml of immune dose, but the serum neutralizing antibody level is not more than 1:512 generally.

In the prior art, it has been disclosed that the titer of a virus can be gradually increased by continuous culture and passaging in sensitive cells of the virus, and a high-titer seneca virus vaccine seed virus can be obtained, for example, in patent document CN110551694A (hereinafter referred to as document 2), SVV/CH/ZZ/2016CGMCC No.14886 is continuously passaged on sensitive cells PK-15 for 12 generations, and the titer of the virus strain can be increased from 10 to 10^6.5TCID₅₀Increase of/ml to 10^7.75TCID₅₀0.1ml, but this method only allows a limited increase in the titer of the virus and does not necessarily increase the pathogenic capacity of the virus in the present animal swine, which may still be insufficient for the preparation of a seneca virus vaccine product with particularly desirable immunological effects. It is therefore desirable to provide a highly virulent seneca virus vaccine seed virusIn order to further enhance the immunopotency of the seneca virus vaccine.

Disclosure of Invention

In view of one or more of the problems in the prior art, one aspect of the present invention provides a method for obtaining a highly virulent seneca virus vaccine seed virus, comprising the steps of:

s1: selecting a diseased tissue with the most severe clinical symptom expression of a diseased pig infected by the Seneca virus original virus, inoculating PK-15 cells after treatment, culturing and passaging until the PK-15 cells can generate obvious and typical cytopathic effect (preferably culturing and passaging until the PK-15 cells can generate obvious and typical cytopathic effect for the first time), and harvesting a virus solution;

s2: carrying out continuous N-generation subvirus cloning and purification on the virus liquid obtained in the step S1 to obtain N seed viruses, wherein N is more than or equal to 1;

s3: detecting the titer of the seed virus obtained after each cloning and purification in the step S2 to obtain a high-titer seneca virus seed virus, wherein the high-titer seneca virus seed virus meets the following requirements: the titer of the seed virus is more than or equal to 10^8.0TCID₅₀/0.1ml；

S4: detecting the pathogenic capability of the high-titer senany virus seed obtained in the step S3 on the healthy pig until the high-toxicity senany virus vaccine seed is obtained;

the high-virulence seneca virus vaccine seed virus meets the following requirements: the titer of the seed virus is more than or equal to 10^8.0TCID₅₀0.1ml, and the disease rate of the seed virus to the animal pig is more than or equal to 80 percent.

In the above method, the high virulence senany vaccine seed further satisfies one or more of the following conditions: the titer of the seed virus is more than or equal to 10^8.25TCID₅₀0.1ml, preferably the titer of the seed virus is more than or equal to 10^8.5TCID₅₀0.1 ml; the disease rate of the seed virus to the animal pig is more than or equal to 90 percent, and the preferable disease rate is 100 percent.

In the above method, the clinical symptoms in step S1 include one or more of the following: blisters, ulcerations or scabs appear on the hoof palm, crown edge or hoof-attached toe; and blisters, ulcerations or crusting on the osculating process; and/or the diseased tissue comprises one or more of: blisters, ulcerations or crusts on the hoof palm, crown edge or toe; and blisters, ulcerations or crusts on the osculating process.

In the above method, the Seneca virus original virus in step S1 is F7 strain of SVV/CH/ZZ/2016CGMCC No. 14886.

In the above method, if the high titer senany virus seed is not obtained in step S3, any one of the N viruses obtained after cloning and purification (preferably, the seed with the highest titer among the N viruses and the lowest generation of cloning and purification) is used to replace the original senany virus in step S1, and steps S1 to S3 are repeated until the high titer senany virus seed is obtained; and/or if the high-virulence senany of the N kinds of viruses obtained after cloning and purification (preferably the kind of virus with the highest titer and the lowest cloning and purification generation number of the N kinds of viruses) is not obtained in the step S4, replacing the original virus of the senany virus in the step S1, and repeating the steps S1 to S4 until the high-virulence senany of the senany vaccine viruses is obtained.

In the method, the obvious and typical cytopathy means that the cytopathy degree is more than 70%, the phenomena of typical cell enlargement and rounding can be seen in the early stage of the cytopathy, the refractivity is enhanced, and then local cells begin to fuse; the late stage cells begin to be fused in a large area and are in a net shape, and finally the fused cells are necrotic and shed.

In the above method, the step S1, after harvesting the virus fluid, further comprises continuously passaging the virus fluid on PK-15 cells for more than 2 generations, and generating stable distinct and typical cytopathic effect on the PK-15 cells.

In the above method, step S1 further comprises the step of performing PCR identification on the diseased tissue and the harvested virus fluid or the virus fluid obtained by continuously passaging the virus fluid on PK-15 cells; optionally, PCR primers used in the PCR identification are shown as SEQ ID NO.1 and SEQ ID NO. 2.

In the method, the treatment in the step S1 is that the diseased tissues of the diseased pigs are washed by 1000-2000 IU/ml streptomycin, cut into pieces, ground, added with PBS containing 100-200 IU/ml each of penicillin and streptomycin sulfate, and soaked overnight; sucking out the soaking solution, centrifuging to obtain supernatant, and filtering with filter membrane (pore diameter of 0.22 μm) for sterilization.

In the above method, the specific operations of culturing and passaging in step S1 are: after being treated, the diseased tissue of the diseased pig is inoculated on a full monolayer of PK-15 cells according to the amount of 5-10% of the volume ratio of a cell culture medium, is adsorbed for 1-2 hours at the temperature of 20-25 ℃, and is placed in CO at the temperature of 37 DEG C₂Continuously culturing in an incubator for passage, observing lesions every day until PK-15 cells can generate obvious and typical cytopathic lesions for the first time, and harvesting virus liquid.

In the above method, the virus clone is purified by the plaque cloning method in step S2;

optionally, the specific operation of purifying the virus clone is as follows: taking the harvested virus liquid for 10-fold serial dilution, respectively inoculating the serial diluted virus liquid into a porous plate full of a single-layer PK-15 cell, setting multiple holes for each dilution, and simultaneously setting a blank control; placing the mixture in a 5% carbon dioxide incubator at 37 ℃ for adsorption for 1 hour, and shaking up the mixture every 15 minutes during adsorption; after adsorption, taking out the porous plate, removing the adsorption solution, adding agar nutrient solution into each hole, standing in a refrigerator at 4-8 ℃ (or at normal temperature) for 5-10 minutes to solidify the agar nutrient solution, taking out and inversely putting into an incubator at 36-37 ℃ and containing 5% of carbon dioxide to culture for 48-72 hours; after the porous plate generates spots, selecting independent spots with proper size, repeatedly blowing and sucking the spots by using a virus maintenance liquid, inoculating the spots into a full monolayer of PK-15 cells, placing the cells in a 37 ℃ and 5% carbon dioxide incubator for adsorption for 1 hour, supplementing the virus maintenance liquid, placing the cells in the 37 ℃ and 5% carbon dioxide incubator for culture, observing pathological changes every day, harvesting the virus liquid when the pathological changes of the cells reach 70% -90%, freezing and thawing for 3 times, and obtaining the virus liquid after cloning and purifying for one generation, which is marked as P1; and (4) continuing to clone and purify the virus liquid P1 according to the operation, wherein the cloning and purification are carried out for N times in total, and N is more than or equal to 1.

In another aspect, the present invention provides a high virulence senany virus vaccine seed obtained by the above method, wherein the high virulence senany virus vaccine seed satisfies: the titer of the seed virus is more than or equal to 10^8.0TCID₅₀0.1ml, and the disease rate of the seed virus to the animal pig is more than or equal to 80 percent;

preferably, the high virulence seneca virus vaccine seed further satisfies one or more of the following conditions: the titer of the seed virus is more than or equal to 10^8.25TCID₅₀0.1ml, preferably the titer of the seed virus is more than or equal to 10^8.5TCID₅₀0.1 ml; the disease rate of the seed virus to the animal pig is more than or equal to 90 percent, and the preferable disease rate is 100 percent.

The application of the high-virulence senany vaccine seed virus in the preparation of the senany vaccine also belongs to the content of the invention.

The method for obtaining the high-toxicity Seneca virus vaccine seed virus provided by the technical proposal selects the diseased tissue with the most serious clinical symptom expression of the sick pig infected by the original Seneca virus (the diseased tissue can be the scab which is necrotized and shed from the most serious diseased part of the diseased pig after the original Seneca virus attacks the healthy and susceptible animal pig), and the clinical symptom expression of the diseased pig is the most serious diseased tissue (the diseased tissue can be the hoof skin, blister skin or scab sample which is obtained from the most serious diseased part of the diseased pig and preserved in the laboratory). And culturing, subculturing and continuously cloning and purifying for multiple times to obtain the high-toxicity epinastine vaccine seed virus, in particular to the epinastine vaccine seed virus with high titer and strong pathogenicity to the animal pig. The results of the examples show that when the F7 strain of SVV/CH/ZZ/2016CGMCC No.14886 was used (the virus titer was 10)^7.0TCID₅₀0.1ml, the pathogenic rate of the animal pig is 40%) as the original virus of the Seneca virus, selecting the separated antigen of the diseased tissue with the most serious clinical symptom expression of the sick pig infected by the original virus of the Seneca virus, culturing and passaging twice in sensitive PK-15 cells, and cloning and purifying twice to obtain the dripping pigDegree of 10^8.0TCID₅₀0.1ml or more (10)^8.25TCID₅₀0.1ml) and has a disease rate of 100% in the swine of the animal, is significantly higher than the titer of the original virus and the disease rate in the swine of the animal, and is also significantly higher than the titer (10) of the vaccine seed virus disclosed in the above-mentioned document 1^7.0TCID₅₀0.1ml) and the morbidity (80%) of the pigs of the animals, and the virus titer is also obviously higher than the virus titer level (10) which is achieved by continuously passaging SVV/CH/ZZ/2016CGMCC No.14886 on sensitive cells PK-15 for 12 generations in the literature 2^7.75TCID₅₀0.1 ml). When the obtained high-virulence Secoid virus vaccine seed virus is prepared into a vaccine for use, the serum neutralizing antibody titer level of 28 days after first immunization is generally over 1:724 and even can reach 1:2048 under the immune dose of 3ml, and the vaccine has a particularly ideal immune protection effect.

Therefore, the method provided by the invention can effectively obtain the seneca virus vaccine seed virus with high toxicity (high titer and strong immunogenicity), and can be used for producing the seneca virus vaccine product with particularly ideal immune effect.

Drawings

FIG. 1 is an electron micrograph of normal cells and cells after inoculation;

FIG. 2 is a gel electrophoresis image of PCR identification of virus liquid.

Detailed Description

Aiming at the defects that the vaccine prepared by the prior art has poor immunity efficacy and can not reach a particularly ideal state due to low titer and weak immunogenicity of the seed virus used in the preparation of the Seneca virus vaccine, the invention provides a method for obtaining the seed virus of the Seneca virus vaccine with high toxicity so as to be beneficial to preparing the Seneca virus vaccine with particularly ideal immunity effect.

The present invention will be described in detail with reference to the following specific embodiments.

The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: a molecular Cloning protocol ("molecular Cloning: A Laboratory Manual," Sambrook, J., Russell, David W., molecular μ Lar Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor ").

The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, which are helpful for understanding the invention, but should not be taken as limiting the content of the invention.

Example 1 challenge of the animal pig with the original Seneca Virus

In this example, F7 generation virus liquid using SVV/CH/ZZ/2016CGMCC No.14886 (see CN110551694A) was selected as the original virus liquid of Seneca virus (the titer of virus was determined to be 10)^7.0TCID₅₀0.1ml, provided by jin Yu Baoling biopharmaceutical Co., Ltd.) to vaccinate domestic pigs for challenge testing, specifically comprising the following steps.

(1) Selecting experimental animals: selecting more than 10 healthy susceptible pigs (purchased from a certain pig farm in Huohahote) with age of more than 90 days, unlimited male and female, and SVA serum neutralizing antibody titer level less than 1: 2. Of these, 5 were used as test groups and the other 5 as controls.

(2) Using original virus liquid of the Seneca virus to carry out virus attack on domestic pigs of a test group, wherein the virus attack way is nose dropping, the virus attack dose is 5.0ml per head, and each nostril is 2.5 ml; the control group was inoculated with physiological saline in the same manner and dosage.

(3) Observing the disease condition of the domestic pigs of the test group every day after the toxin attack, and finding that the domestic pigs of the test group begin to have blisters, ulcerations or scabs on the hoof palm part, the edge of the hoof crown part or the hoof-attached toe part on the 5 th day after the toxin attack; the rhynchophorus part has clinical symptoms such as blister, ulceration or scabbing, the incidence rate is 2/5, and the control group pigs have no abnormality. The diseased tissue of the diseased pig was subjected to PCR detection, and the result was positive for SVA (the specific PCR detection method is described in example 2 below). The result of the challenge test shows that the F7 virus liquid of SVV/CH/ZZ/2016CGMCC No.14886 as the original virus has weak pathogenic ability to the pig and the pathogenic rate only reaches 40 percent, which indicates that the vaccine has weak immunogenicity and is difficult to prepare the Seneca virus vaccine product with particularly ideal immune effect when being used as the seed virus of the Seneca virus vaccine.

Example 2 obtaining of high-virulent Ex Canava vaccine seed Virus

Taking the diseased tissue with the most severe clinical symptom expression (namely the most obvious clinical symptom expression) with the SVA positive detection result of the PCR detection result of the diseased pig in the example 1, and inoculating PK-15 adherent cells after treatment.

(21) And (3) treatment of pathological materials: selecting the diseased tissue (hoof skin, blister skin or scab) with the most serious clinical symptom expression of the diseased pig, flushing the diseased tissue with 2000IU/ml streptomycin, shearing, grinding, adding a proper amount of PBS containing 100IU/ml each of penicillin and streptomycin sulfate, and soaking overnight. Sucking out the soaking solution, centrifuging at 5000r/min for 10 min, collecting supernatant, and filtering with filter membrane with pore diameter of 0.22 μm for sterilization.

(22) Inoculating cells: inoculating the supernatant treated in the step (21) on a full monolayer of PK-15 cells according to the volume ratio of 5-10% of a cell culture medium (DMEM), adsorbing for 1-2 hours at 20-25 ℃, and placing in CO at 37 DEG C₂Continuously culturing in incubator, observing cytopathic effect (CPE) every day, culturing for 24-72 hr or CPE above 70%, harvesting virus solution, repeatedly freezing and thawing for 3 times, labeled as F1, and storing in refrigerator at-20 deg.C. The harvested virus fluid F1 was further inoculated with a monolayer of PK-15 cells for passage according to the method described above, and the virus passage was found to be until the virus F2 began to present CPE and was able to cause obvious and typical cytopathic effects on the PK-15 cells, and the harvested virus fluid was named F2. After the continuous 3 generations (the harvested virus liquid is respectively named as F3, F4 and F5) according to the operation, stable obvious and typical regular cytopathic effect can be generated within 24-48 hours (the purpose of continuous passage is to prove that the cytopathic effect is really caused by some pathogenic microorganisms, and the continuous stable passage can be performed on cells). Wherein is obvious and typicalThe cytopathic effect of (a) is as follows: the degree of cytopathic effect reaches more than 70%, the typical phenomena of cell enlargement and cell rounding can be seen in the early stage of the effect, the refractivity is enhanced, and then local cells begin to fuse; the late stage cells begin to be fused in a large area and are in a net shape, and finally the fused cells are necrotic and shed. The cells of the control group (uninoculated diseased tissue) were not abnormal. The growth state of the cells before virus inoculation, the growth state of the cells in the control group and the growth state of the cells after virus inoculation are shown in figure 1, and it can be seen that the growth states of the cells before virus inoculation and the cells in the control group are good, no abnormal cytopathic condition occurs, and the cells after virus inoculation have obvious and typical cytopathic effect.

(23) And (3) nucleic acid detection: in this step, virus nucleic acid was detected by using the virus solution passaged to F4 in step (22) (F4 virus solution). Specifically, RNA extraction (adopting an RNA extraction kit purchased from Biotechnology engineering Co., Ltd.) is carried out on the virus liquid harvested from the F4 generation, and the specific operation method comprises the following steps: adding 200 mu L F4 virus solution into a 1.5ml centrifuge tube, adding 200 mu L Buffer V-L, mixing uniformly by vortex oscillation, and standing for 5 minutes; adding 75 mul Buffer V-N, mixing evenly by vortex oscillation, and centrifuging for 5 minutes at 12000 rpm; transferring the supernatant into a new 2ml centrifuge tube, adding 300 mu l of isopropanol (1% glacial acetic acid), inverting for 6-8 times, mixing uniformly, transferring the mixture into a preparation tube, placing the preparation tube into the 2ml centrifuge tube, and centrifuging at 6000rpm/min for 1 minute; discarding the filtrate, placing the preparation tube back into a 2ml centrifuge tube, adding 500. mu.l Buffer W1A, placing the tube in a greenhouse for standing for 1 minute, and centrifuging at 12000rpm for 1 minute; discarding the filtrate, placing the preparation tube back into a 2ml centrifuge tube, adding 800. mu.l Buffer W2, and centrifuging at 12000rpm for 1 minute; discarding the filtrate, putting the preparation tube back into a 2ml centrifuge tube, and centrifuging at 12000rpm for 1 minute; the prepared tube is placed in a clean 1.5ml centrifuge tube, 40 mul of enzyme-free water is added in the center of the prepared tube membrane, the mixture is kept stand for 1 minute at room temperature, and 12000g of the mixture is centrifuged for 1 minute to obtain RNA. The specific operation method for identifying SVA by using PCR method is as follows: the PCR primers used included the forward primer SVA-F: 5'-TATCTCAGATCCCTGGCTGTC-3' (SEQ ID NO:1), downstream primer SVA-R: 5'-CCTGATGATCACATTGTTGAGC-3' (SEQ ID NO:2), and the fragment size of the amplification product is 129 bp. Reaction system: 2 × One Step RT-PCR Buffer III 12.5 μ l, TaKaRa Ex Taq HS0.5. mu.l, 0.5. mu.l of PrimeScript RT Enzyme Mix II, 0.5. mu.l of SVA-F (20. mu.M), 0.5. mu.l of SVA-R (20. mu.M), 2.0. mu.l of RNA template, RNA-free H₂O5.8. mu.l. The PCR reaction conditions are as follows: firstly, reverse transcription is carried out for 15 minutes at 42 ℃, and pre-denaturation is carried out for 2 minutes at 95 ℃; then denatured at 94 ℃ for 10 seconds, annealed at 59 ℃ for 30 seconds, and subjected to 45 cycles (PCR amplification). The PCR amplification product was subjected to 1.5% agarose gel electrophoresis, and the result was observed with a gel imager, as shown in fig. 2, it was found that a bright single band appeared around 129bp, which was consistent with the size of the target fragment amplified by the PCR primer and the size of the amplified fragment of the positive control (shown in lane P in fig. 2), and it was confirmed that the F4 virus solution was seneca virus solution, and it was also confirmed that the F2 virus solution from which the F4 virus solution was propagated was seneca virus solution.

(24) Cloning and purification of the virus: the success rate of the method of the present invention can be improved by the passage of step (22) to F2 (F2 is the passage of the virus fluid with obvious and typical cytopathic effect for the first time, and the virus fluid with the lowest passage is used for clone purification, although the virus concentration in the virus fluid with the highest passage is higher than that in the virus fluid with the lowest passage, some non-target viruses may adapt to cells gradually along with the passage of the target viruses and may generate lesions, therefore, the virus fluid with the second passage of the obvious and typical cytopathic effect for the first time is preferably subjected to clone purification by 10-fold serial dilution to 10 times in the method of the present invention)^-4、10^-5、10^-6Respectively inoculating the cells into 6-hole plates full of monolayer PK-15 cells, making 4 holes for each dilution, inoculating 200 mu l of each hole, and simultaneously setting 2 holes for blank control; placing the mixture in a 5% carbon dioxide incubator at 37 ℃ for adsorption for 1 hour, and shaking up the mixture every 15 minutes during adsorption. And (3) after adsorption, taking out the 6-hole plate, removing the adsorption solution, adding 2ml of agar nutrient solution into each hole, standing for 5-10 minutes in a refrigerator at 4-8 ℃ (or at normal temperature) to solidify the agar nutrient solution, taking out, inverting, and putting into an incubator at 36-37 ℃ and containing 5% of carbon dioxide for culturing for 48-72 hours. After 6-well plate spots appeared, the results are shown in Table 1 below, selecting independent spots with appropriate size, repeatedly sucking with 1ml virus-holding solution, inoculating into T25 full of monolayer PK-15 cells (equivalent to 10% inoculation), placing in a 5% carbon dioxide incubator at 37 deg.C for 1 hr, and supplementing with diseaseCulturing the virus maintaining liquid in a 37 ℃ 5% carbon dioxide incubator, setting cell contrast, observing pathological change conditions every day, harvesting virus liquid when the pathological change of cells reaches 70% -90%, freeze thawing for 3 times, mixing the virus liquid and subpackaging in 1.5ml centrifugal tubes, wherein 1ml of each tube is marked as F21. F21 was further subjected to clone purification passage according to the above method for 13 passages (i.e., N ═ 13), and 13 clone-purified seneca virus strains, labeled F21-F213, were harvested.

Table 1: clonal viral plaque count

Note: the number of plaques is the total number of plaques present per 4 wells of dilution;

"/" indicates not counted.

(25) The titer of the seed virus after clone purification was determined and compared with the original virus: diluting various virus solutions after cloning and purifying with virus maintaining solution 10 times, and taking 10 times^-5、10^-6、10^-7、10^-84 dilutions, each dilution is inoculated to a 96-well cell culture plate with 100 mul per well, each dilution is inoculated to 4 wells, then 100 mul of cell fluid is supplemented to each well, meanwhile, a normal cell control with 4-8 wells and 100 mul of dilution per well are set, then 100 mul of PK-15 cell suspension is supplemented to each well, the temperature is set at 37 ℃, and 5% CO is contained₂Culturing for 48-72 hours in an incubator, observing cytopathic effect under a microscope, recording the number of cytopathic holes of each dilution, and calculating TCID according to Reed-Muench method₅₀. As a result, as shown in Table 2 below, it was found that the titer of F21 was 10^7.0TCID₅₀0.1ml, the titers of F22-F213 were all 10^8.0TCID₅₀More than 0.1ml (even up to 10)^8.5TCID₅₀0.1ml), namely the titer of the seed virus obtained after 2 generations of clone purification is higher than that of the original Seneca virus (10)^7.0TCID₅₀0.1ml) was increased by about 1 to 1.5 titers (10 to 30 times), and was also significantly higher than the titer (10) of the seed virus for vaccine disclosed in the above-mentioned document 1^7.0TCID₅₀0.1ml), and SVV/CH/ZZ/2016CGMCC No.14886 was sensitized in the above-mentioned document 2Virus titer levels achieved by 12 serial passages on cell PK-15 (10)^7.75TCID₅₀/0.1ml)。

Table 2: detection results of various titer of viruses after cloning and purification

(26) Verifying the seed virus after cloning and purification: respectively extracting F26 and F213 virus liquid nucleic acids according to the step (23), and respectively carrying out PCR detection on Seneca virus, foot and mouth disease virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, pseudorabies virus, porcine transmissible gastroenteritis virus and porcine diarrhea virus. The identification result is positive for Seneca virus, and the other detection items are negative.

(27) The pathogenic capability of the cloned and purified seed virus to the pig of the animal is determined: taking 5 pigs of 90-95 days old, dripping 5ml of cloned and purified F22 seed toxin into the nose of each pig, and continuously observing for 10 days after toxin attack to obtain a toxin attack group, wherein the left nostril and the right nostril respectively have 2.5 ml; 2 pigs of 90-95 days old are taken, each pig is dripped into the nose to attack 5ml PBS, the left nostril and the right nostril are respectively 2.5ml, and the pigs are continuously observed for 10 days to serve as a control group. As shown in table 3 below, it can be seen that 5 pigs in the challenge group all exhibited the typical symptoms of SVA infection, the disease rate was 5/5, which was significantly higher than the challenge disease rate (40%) of the original virus and significantly higher than the challenge disease rate (80%) of the vaccine seed virus disclosed in the above document 1, and the control group 2 pigs were not abnormal. Therefore, the F22 virus can be used as a high-virulence Semliki virus vaccine virus.

Table 2: the result of the determination of the pathogenic capability of the cloned and purified seed virus on the animal pig

The results of the above example 2 show that the method for obtaining a high-virulence senany vaccine seed virus of senany virus selects the pathogenic tissue with the most severe clinical symptom expression of the sick pig infected by the original senany virus (the pathogenic tissue can be the pathogenic tissue which can cause the attacked pig to generate the typical clinical symptoms after the original senany virus attacks the healthy and susceptible animal pig, and the pathogenic tissue with the most severe clinical symptom expression. after the original senany virus attacks the animal pig, the breeding can be performed for a plurality of rounds in the pig body, so that the virulence of the senany virus can be improved, and the virulence of the senany virus existing in the pathogenic tissue with the most severe clinical symptom expression is stronger), and the breeding method is subjected to culture passage and continuous cloning and purification for a plurality of times, can obtain high-toxicity epinastine vaccine seed virus, in particular to the epinastine vaccine seed virus which has high titer and strong pathogenic capability to the pig of the animal.

This example used a F7 strain of SVV/CH/ZZ/2016CGMCC No.14886 (virus titer 10)^7.0TCID₅₀0.1ml, the pathogenic rate of the animal pig is 40%) as the original virus of the Seneca virus, selecting the separated antigen of the diseased tissue with the most serious clinical symptom expression of the sick pig infected by the original virus of the Seneca virus, culturing and passaging twice in sensitive PK-15 cells, and cloning and purifying twice to obtain the titer of 10^8.0TCID₅₀0.1ml or more (10)^8.25TCID₅₀0.1ml) and has a disease rate of 100% for the present animal pigs (i.e. the seneca virus vaccine strains which have obtained the desired high virulence in the first round of operation). However, if the desired high virulence senany of the seniviruses of the Sagnac virus vaccine strains is not obtained after the clone purification of the first round of operation, it is necessary to repeat one or more of the operations (i.e. performing the second round of operation, the third round of operation, etc.) from steps (21) to (27) of example 2 until the desired high virulence senany of the seniaviruses of the Segnac virus vaccine strains is obtained, wherein in each round of operation, any one of the N (N.gtoreq.1) strains obtained after the clone purification of the previous round of operation can be selected to replace the original one of the seniaviruses of the next round of operation, but preferably the one with the highest titer among the N strains and the lowest clone purification number of generations is used to replace the next one of the next round of operationThe original virus of the seneca virus is obtained by the rotation operation, so that the seed virus of the needed seneca virus with high toxicity can be obtained quickly.

Therefore, the method for obtaining the high-virulence senany virus vaccine seed virus provided by the invention can comprise the following steps:

s1: selecting a diseased tissue with the most severe clinical symptom expression of a diseased pig infected by the Seneca virus original virus, inoculating PK-15 cells after treatment, culturing and passaging until the PK-15 cells can generate obvious and typical cytopathic effect (preferably, the PK-15 cells can generate obvious and typical cytopathic effect for the first time), and harvesting a virus solution;

s3: the titer of the seed virus obtained after each cloning and purification in the step S2 is detected to obtain the high-titer seneca virus seed virus (the titer is more than or equal to 10)^8.0TCID₅₀0.1 ml); if the high-titer senany virus seed virus is not obtained in the step, any one of N seed viruses obtained after cloning and purification (preferably the seed virus with the highest titer and the lowest cloning and purification generation number in the N seed viruses) is used for replacing the original senany virus in the step S1, and the steps S1-S3 are repeated until the high-titer senany virus seed virus is obtained;

s4: the pathogenic capability of the high-titer seneca virus seed virus obtained in the step S3 on the pig of the animal is detected until the high-virulence seneca virus vaccine seed virus is obtained (the titer is more than or equal to 10 is satisfied)^8.0TCID₅₀0.1ml, and the disease rate of the seed virus to the animal pig is more than or equal to 80 percent; preferably one or more of the following conditions are also met: the titer of the seed virus is more than or equal to 10^8.25TCID₅₀0.1ml, preferably the titer of the seed virus is more than or equal to 10^8.5TCID₅₀0.1 ml; the disease rate of the seed virus to the animal pig is more than or equal to 90 percent, and the preferable disease rate is 100 percent); if the step does not obtain the high-toxicity Seneca virus vaccine seed virus, any one of the N seed viruses obtained after cloning and purifying (preferably the seed virus with the highest titer and the lowest cloning and purifying generation) is used for replacing the original Seneca virus in the step S1, and the step is repeatedS1-S4 until a highly virulent Seneca virus vaccine seed is obtained.

Example 3: efficacy evaluation of Selenecar virus vaccines prepared from high-virulence Selenecar virus vaccine seed virus

This example used the F22 virus fluid obtained in example 2 above (titer 10)^8.25TCID₅₀0.1ml) as a seed virus of the Seneca virus vaccine, the inactivated vaccine was prepared by the method described in example 2 of the above document 1 and by the method described in test example 3 of the above document 1, and the serological potency test was carried out on the prepared inactivated vaccine, and the test results are shown in Table 4 below. As can be seen, the serum neutralizing antibody titer level of the inactivated vaccine prepared by using the F22 virus liquid as the vaccine seed virus is generally over 1:724 and can be as high as 1:2048 after the first immunization in 3ml of immune dose, which proves that the F22 virus inactivated vaccine has strong immunogenicity, and the prepared Seneca virus inactivated vaccine has excellent immune protection efficacy.

Table 4: neutralizing antibody titer levels for serological potency testing of inactivated vaccines

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the invention as defined by the appended claims. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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Claims

1. A method for obtaining a highly virulent seneca virus vaccine seed virus, comprising the steps of:

the high-virulence seneca virus vaccine seed virus meets the following requirements: the titer of the seed virus is more than or equal to 10^8.0TCID₅₀0.1ml, and the disease rate of the seed virus to the pig is more than or equal to 80 percent.

2. The method of claim 1A method, said high virulence seneca virus vaccine seed further satisfying one or more of the following conditions: the titer of the seed virus is more than or equal to 10^8.25TCID₅₀0.1ml, preferably the titer of the seed virus is more than or equal to 10^8.5TCID₅₀0.1 ml; the disease rate of the seed virus to the pig is more than or equal to 90 percent, and the preferable disease rate is 100 percent.

3. The method of claim 1 or 2, wherein the clinical symptoms in step S1 include one or more of: blisters, ulcerations or scabs appear on the hoof palm, crown edge or hoof-attached toe; and blisters, ulcerations or crusting on the osculating process; and/or

The diseased tissue includes one or more of: blisters, ulcerations or crusts on the hoof palm, crown edge or toe; and blisters, ulcerations or crusts on the osculating process.

4. The method according to any one of claims 1 to 3, wherein the seneca virus primary virus in step S1 is F7 strain of SVV/CH/ZZ/2016CGMCC No. 14886.

5. The method according to any one of claims 1 to 4, wherein if no high titer Selenavirus seed virus is obtained in step S3, any one of the N seed viruses obtained after clonal purification (preferably the seed virus with the highest titer of the N seed viruses and the lowest passage of clonal purification) is used instead of the original Selenavirus seed virus in step S1, and steps S1 to S3 are repeated until a high titer Selenavirus seed virus is obtained; and/or

If the high-virulence senany of the S4 vaccines is not obtained, any one of the N viruses (preferably the one with the highest titer and the lowest cloning and purification generation number) obtained after cloning and purification is used to replace the original senivirus in the S1, and the steps S1 to S4 are repeated until the high-virulence senivirus vaccine is obtained.

6. The method according to any one of claims 1-5, wherein the overt and typical cytopathy is a cytopathy of more than 70%, typical cell enlargement, rounding, increased refractivity, and then local cell fusion; the late stage cells begin to be fused in a large area and are in a net shape, and finally the fused cells are necrotic and shed.

7. The method of any one of claims 1 to 6, further comprising, after harvesting the viral fluid, continuing serial passage of the viral fluid on the PK-15 cells for more than 2 generations and causing stable, distinct and typical cytopathic effects on the PK-15 cells in step S1; and/or

Step S1 also includes the step of PCR identification of the pathogenic tissue and the harvested virus liquid or the virus liquid obtained by continuous passage of the virus liquid on PK-15 cells; optionally, PCR primers used in the PCR identification are shown as SEQ ID NO.1 and SEQ ID NO. 2; and/or

In the step S1, the diseased tissue of the diseased pig is washed by 1000-2000 IU/ml streptomycin, cut into pieces, ground, added with PBS containing 100-200 IU/ml each of penicillin and streptomycin sulfate, and soaked overnight; sucking out the soaking solution, centrifuging to obtain supernatant, and filtering with filter membrane (pore diameter of 0.22 μm) for sterilization; and/or

The specific operations of culturing and subculturing in the step S1 are as follows: after being treated, the diseased tissue of the diseased pig is inoculated on a full monolayer of PK-15 cells according to the amount of 5-10% of the volume ratio of a cell culture medium, is adsorbed for 1-2 hours at the temperature of 20-25 ℃, and is placed in CO at the temperature of 37 DEG C₂Continuously culturing in an incubator for passage, observing lesions every day until PK-15 cells can generate obvious and typical cytopathic lesions for the first time, and harvesting virus liquid.

8. The method of any one of claims 1-7, wherein the viral clone is purified in step S2 using a plaque cloning method;

9. A high virulence senany vaccine seed obtained by the method of any one of claims 1-8, wherein the high virulence senany vaccine seed satisfies: the titer of the seed virus is more than or equal to 10^8.0TCID₅₀0.1ml, and the disease rate of the seed virus to the pig is more than or equal to 80 percent;

preferably, the high virulence seneca virus vaccine seed further satisfies one or more of the following conditions: the titer of the seed virus is more than or equal to 10^8.25TCID₅₀0.1ml, preferably the titer of the seed virus is more than or equal to 10^8.5TCID₅₀0.1 ml; the disease rate of the seed virus to the pig is more than or equal to 90 percent, and the preferable disease rate is 100 percent.

10. Use of the high-virulence seneca virus vaccine seed virus of claim 9 for the preparation of a seneca virus vaccine.