CN111876391A - Feline panleukopenia virus FPV BJ05 strain and application thereof - Google Patents

Abstract

The present invention provides feline panleukopenia virus FPV BJ05 strain and application thereof. The strain FPV BJ05 has the advantages of good immunogenicity, good culture characteristics and stable titer. The FPV BJ05 strain was infectiously cloned to obtain a genetically marked rescue strain FPV BJ05C, and the rescue strain was inoculated into susceptible cells and harvested. The culture is inactivated with β-propiolactone, mixed with aluminum hydroxide adjuvant and emulsified to prepare an inactivated feline panleukopenia virus vaccine. Experiments show that using the feline panleukopenia virus inactivated vaccine provided by the present invention to immunize healthy kittens after weaning can prevent diseases caused by FPV, and the vaccine has the advantages of safety, fast onset, and long-lasting immunity.

Description

Feline panleukopenia virus FPV BJ05 strain and its application

technical field

The invention relates to the field of biological products and preventive veterinary medicine, in particular to the feline panleukopenia virus FPVBJ05 strain and its application.

Background technique

Feline panleukopenia virus is an unencapsulated, single-stranded DNA virus that is highly contagious and lethal to cats and felines. Can cause cat and feline panleukopenia (Feline panleukopeniadisease, FPD). The virus is trending worldwide and is considered to be the most widespread and most pathogenic virus of carnivore parvovirus. The virus was first discovered in 1928 and officially named Feline Panleukopenia Virus in 1935. FPV is transmitted by the fecal-oral route, mainly by contact with infected body fluids, feces or other pollutants, and by fleas. FPV is highly resistant to the environment and can survive for at least a year in infected tissue samples. Pollutants are a major source of transmission, with cat owners transmitting highly contagious viruses to cats indoors through shoes or clothing. 18 to 24 hours after infection through the nose or mouth, the virus begins to replicate in the oropharynx, viremia occurs after 2 to 7 days, and virus replication mainly occurs in mitotic tissues. The mucous membranes are most commonly affected.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a feline panleukopenia virus FPV BJ05 strain with good immunogenicity, good culture characteristics and stable titer.

Another object of the present invention is to provide the application of the rescued strain of feline panleukopenia virus FPV BJ05 strain (FPVBJ05C strain) in the preparation of vaccines.

In order to achieve the object of the present invention, in a first aspect, the present invention provides Felinepanleukopenia virus FPV BJ05 strain. The inventor isolated a feline panleukopenia virus from an animal hospital in Beijing in 2017, and obtained it from the feces of sick cats with vomiting, diarrhea and severe hemorrhagic enteritis. It was identified as a virulent strain, and the domesticated strain was named as FPV BJ05 strain. Compared with other isolated strains, its proliferation titer on cells was high and its immunogenicity to cats was good. The rescue strain FPV BJ05C strain (vaccine strain) of FPV BJ05 strain was obtained by infectious cloning. Compared with the wild strain (FPV BJ05 strain), a genetic marker was added by site-directed mutation, that is, the 2980-2985 bp of the FPV BJ05 genome was mutated from CTCAAG to CTCGAG. It was verified that the rescued strain had the same biological characteristics as the wild strain. , after passage by cat kidney cell line (F81) as a vaccine virus, the virulent strain used for titer detection is the 7th-10th generation of the wild strain FPV BJ05. This strain can cause more than 90% infection of F81 cells. The genome sequence of FPV BJ05 strain is shown in SEQ ID NO:1.

Feline panleukopenia virus (Feline panleukopenia virus) FPV BJ05 strain has been deposited in the General Microbiology Center of the China Microbial Culture Collection and Management Committee, address No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101, deposit number CGMCC No. 19988, deposit date May 28, 2020.

In the second aspect, the present invention provides any following application of the rescued strain (FPVBJ05C strain) of the feline panleukopenia virus FPV BJ05 strain:

1) for the preparation of vaccines;

2) A diagnostic reagent for preparing feline panleukopenia virus infection and related diseases caused by its infection.

In a third aspect, the present invention provides a composition comprising an inactivated FPV BJ05C strain and a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention provides an immunogenic composition comprising the above composition.

In a fifth aspect, the present invention provides a vaccine composition comprising the above immunogenic composition.

In a sixth aspect, the present invention provides a method for preparing a feline panleukopenia virus inactivated vaccine, comprising the following steps:

(1) Infectious cloning of feline panleukopenia virus FPV BJ05 strain is carried out, and the obtained rescue virus strain is inoculated into susceptible cells for culture to obtain virus liquid;

(2) inactivating the harvested virus liquid with β-propiolactone;

(3) Mix the inactivated virus liquid with the adjuvant to get it.

The aforementioned method, step (1) comprises: bioinformatics analysis of the Y-shaped structure and U-shaped structure at the 3' and 5' ends of the virus, amplifying the feline panleukopenia virus FPV BJ05 strain genome sequence (SEQ ID NO: 1), Insert pBlueScript II (+) vector, construct reverse genetics platform, transfect F81 cells, carry out virus rescue, obtain rescued strains, inoculate into susceptible cells (such as F81 cells) for culture, and obtain virus liquid.

Preferably, the adjuvant in step (3) is aluminum adjuvant, preferably aluminum hydroxide adjuvant (alum adjuvant).

In the aforementioned method, step (2) includes: inactivating the harvested virus liquid with β-propiolactone with a final concentration of 0.02% to 0.05% at 4 to 10° C. for 36 to 48 hours, stirring once every 2 hours during the period, and inactivating the virus solution. The virus solution was hydrolyzed at 37°C for 1 to 2 hours after live. Preferably, the harvested virus liquid is inactivated by β-propiolactone with a final concentration of 0.03% at 4°C for 48h, stirring every 2h during the period, and the virus liquid is hydrolyzed at 37°C for 2h after inactivation. After inactivation, the virus solution was stored at 2-8°C.

Preferably, the feline panleukopenia virus inactivated vaccine is feline panleukopenia virus (FPV BJ05C) inactivated by aluminum hydroxide adjuvant with a final concentration of 1000 μg/ml and β-propiolactone with a final concentration of 0.03%. Co., Ltd.) obtained after emulsification.

The emulsification process adopts the commercialized aluminum hydroxide adjuvant of Thermo Fisher Scientific as the emulsification process. After the adjuvant is sterilized, it can be directly used for the emulsification preparation of the vaccine; before the adjuvant is used, the aluminum adjuvant bottle is gently shaken. Before emulsification, it will be thawed and mixed with the inactivated virus liquid of the same batch, and the aluminum hydroxide adjuvant will be dropped into the virus liquid under aseptic conditions. The volume ratio of adjuvant and virus liquid is 1:3. Mix well for min. In order to stabilize the interface and eliminate air bubbles and obtain the best emulsification effect, it needs to stand for about 30 minutes before dispensing.

In the seventh aspect, the present invention provides the vaccine composition or the feline panleukopenia virus inactivated vaccine prepared according to the above method in the preparation of biological products for treating or preventing feline panleukopenia virus infection and related diseases caused by its infection. applications in .

The disease is characterized by sudden bidirectional fever, vomiting, diarrhea, dehydration, marked leukopenia, and hemorrhagic enteritis in sick cats.

The rescue strain FPV BJ05C strain of feline panleukopenia virus FPV BJ05 strain provided by the present invention has the advantages of good immunogenicity, good culture characteristics, stable titer and the like, and the rescue strain is added with genetic markers, which is convenient for distinguishing wild virus strains from Vaccine strain, inoculate susceptible cells with FPV BJ05C strain, harvest the culture, inactivate with β-propiolactone, mix and emulsify with aluminum hydroxide adjuvant to prepare feline panleukopenia virus inactivated vaccine. Experiments show that using the feline panleukopenia virus inactivated vaccine provided by the present invention to immunize healthy kittens after weaning can prevent diseases including FPD caused by FPV, and the vaccine has the advantages of safety, fast onset, long-lasting immunity, etc. advantage.

Description of drawings

Figure 1 shows the cytopathic effect caused by inoculation of F81 cells with feline panleukopenia virus FPV BJ05C strain in a preferred embodiment of the present invention. Among them, A: cytopathic 72h after inoculation; B: normal cells as negative control.

Figure 2 shows the virus particles of the strain FPV BJ05C observed under a transmission electron microscope in a preferred embodiment of the present invention.

Fig. 3 is the result of indirect immunofluorescence test of feline panleukopenia virus FPV BJ05C strain in a preferred embodiment of the present invention. Among them, A: Fluorescence detection results of FPV BJ05C strain after culturing on F81 cells for 24 hours; B: Fluorescence detection results of normal control cells.

Detailed ways

The present invention provides feline panleukopenia virus FPV BJ05 strain and application thereof. The inventor isolated a porcine parvovirus from an animal hospital in Beijing in 2017, which was identified as a virulent strain, and the domesticated strain was named FPV BJ05 strain. The FPV BJ05 strain was used to rescue the virus through reverse genetic manipulation, and the rescued strain FPV BJ05C was obtained. The research and development of the inactivated feline panleukopenia virus vaccine was carried out, and the feline panleukopenia virus with good immunogenicity and stable titer was rescued. Leukopenia virus FPV BJ05C strain, the most suitable F81 cells for the virus to multiply, and the inactivating agent, adjuvant and other conditions that meet the production of vaccine were selected. The production process, safety, protection rate, immunization procedure, minimum dose, antibody duration and storage period of biological products were tested, and a large number of experimental data were obtained, proving that the vaccine is safe and effective. On this basis, the pilot production was carried out, and after sampling inspection, all of them met the proposed quality standards, and the pilot product was tested for safety and efficacy. The results confirmed that this vaccine can effectively prevent feline panleukopenia virus. Feline panleukopenia caused by feline panleukopenia, on the basis of laboratory tests and intermediate trial production, the present invention has developed a cat panleukopenia syndrome virus (FPV BJ05C strain) inactivated vaccine, and the results show that the vaccine can effectively prevent cat Panleukopenia in cats caused by panleukopenia virus further verifies the quality standards of this vaccine.

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities.

The cat breed used in the following examples is Chinese pastoral cat.

Example 1 Culture and identification of feline panleukopenia virus FPV BJ05C strain

1. Source and standard of poisonous seeds

According to the application requirements of new biological products, combined with a large number of experimental data obtained by the present invention, with reference to "Chinese Veterinary Pharmacopoeia" (2005 edition), the virus seeds for seedling production were identified.

1.1 Sources of poisonous seeds

The virus species used to make this biological product is the FPV BJ05C strain rescued by the feline panleukopenia virus FPV BJ05 through the constructed infectious cloning platform. The FPV BJ05 strain was obtained by the inventor after isolation and domestication from the feces of affected cats in 2017. This strain was isolated from the feces of cats infected with typical feline panleukopenia virus, and compared with other isolated strains, the proliferation titer on cells was high, and the immunogenicity to cats was good. Therefore, it was selected for the construction of an infectious clone for reverse genetic manipulation platform, transfected into F81 cells for strain rescue, and obtained a virus seed (FPV BJ05C) for vaccine production, and biological identification of the virus seed was carried out. The virulent test used was the 7th-10th generation of FPV BJ05 strain. The virus species is the 7th generation virus strain after passage on F81 cells.

1.2 Virus species standard

1.2.1 Hemagglutination valence

Take a 96-well U-shaped micro-reaction plate, add 25 μl PBS (0.01mol/L, pH 7.0-7.4) to each well with a pipette, add 25 μl antigen to each well in the first column, and then dilute the antigen by doubling until Discard 25 μl from well 11. Add 25 μl of PBS to each well, then add 25 μl of 1% porcine red blood cell suspension, mix well with a micro shaker, and act at room temperature for 1 hour, or in a 4°C refrigerator for 2 hours. When judging the results, the highest dilution of the antigen with 50% hemagglutination was used as the judgment end point. The results showed that the agglutination value of the virus species to porcine red blood cells should not be lower than 1:256.

1.2.2 Virus content

The virus seeds were serially diluted 10 times in DMEM medium, 100 μl of each dilution was added to 96-well cell culture plate, and each dilution was repeated 8 times, and then F81 cell suspension was added, 100 μl per well (cell density). 2 × 10 ⁵ /mL, DMEM containing 10% newborn calf serum), and normal cell culture was set as a control, cultured in a 37°C, 5% CO ₂ incubator, and replaced with 2% newborn calf serum after 12 h. In the maintenance solution, continue to observe for 4 to 5 days under the same culture conditions, and observe cytopathic changes (CPE) day by day. Cells pyknosis, aggregate, and increase in granules. Some cells fuse, fall off, and have many voids, which are judged as CPE. The number of wells with cytopathic changes was recorded, and the TCID ₅₀ was calculated according to the Reed-Muench method. The results showed that the viral liquid was not less than 10 ^6.0 TCID ₅₀ per milliliter.

1.2.3 Virulence

Feline panleukopenia virus FPV BJ05C was inoculated into F81 cells, and the virus generation was the 7th-10th generation cytotoxicity of FPV BJ05C; the challenge dose was that the virus content per milliliter should not be less than 10 ^6.0 TCID ₅₀ ; the procedure was intramuscular injection of 45~ There were 5 60-day-old cats, 2ml/cat, and 5 control cats were set up at the same time. Determination criteria are depression, loss of appetite or abstinence; diarrhea, mucus or blood in stool; stool (1:5 dilution) hemagglutination titer is not less than 1:64, and the hemagglutination performance is confirmed by feline panleukopenia virus Positive serum inhibition.

1.2.4 Immunogenicity

The virus seeds were synchronously inoculated into F81 cells for proliferation, and the virus solution was harvested, inactivated with β-propiolactone with a final concentration of 0.03%, and then added with aluminum hydroxide adjuvant to a final concentration of 800ug/ml, and mixed evenly. 10 healthy susceptible cats aged 45-60 days (FPV HI antibody titer not higher than 1:8) were randomly divided into 2 groups with 5 cats in each group. One group was injected subcutaneously with 1ml of the prepared vaccine, and the other group was used as a control, not vaccinated. Feed in isolation under the same conditions. After 21 days, blood was collected, serum was collected, and HI titer was determined; at the same time, all cats were subcutaneously injected with the virulent culture medium of the 7th-10th generation of FPV BJ05 strain (10 ^6.0 TCID ₅₀ /ml) 1m1, and anal swabs were collected every day. , and were observed for 21 consecutive days. All 5 cats in the control group should have the disease, and the judgment criteria were: depression, loss of appetite or abstinence; diarrhea, mucus or blood in the feces; less than 1:64, and hemagglutination was inhibited by feline panleukopenia virus-positive serum. The HI antibody titer should not be higher than 1:8; at least 4 of the 5 cats immunized should be alive and healthy, and the HI antibody titer should not be lower than 1:32.

1.2.5 Purity test

Bacterial, mold and mycoplasma tests were carried out on the 1st to 5th generations of the original seed batch, the 6th to 15th generations of the basic seed batch and the 16th to 18th generations of the production seed batch of the established feline panleukopenia virus FPV BJ05C strain. The 3rd, 9th and 17th generation virus seeds have been tested for exogenous viruses, and the results show that the original seed batches, basic seed batches and production seed batches established by the present invention are free from bacterial, mold, mycoplasma and exogenous virus contamination. kind of pure.

1.2.6 Specificity

Dilute the virus seed to 200TCID ₅₀ /0.1ml with DMEM cell culture medium, mix it with an equal amount of inactivated anti-feline panleukopenia virus-specific serum, and incubate it in a 37°C water bath for 1h. Simultaneous inoculation grows well. 3 flasks of F81 cells (the cell density was 2×10 ⁵ /ml), and the virus control, normal cell control and negative serum control were set at the same time, and were placed in a 37°C, 5% CO ₂ incubator for 5 to 7 days. There should be no cytopathic changes (CPE) in the neutralized virus group and the normal cell control group; CPE should appear in both the virus control group and the negative serum control group.

1.2.7 Generations of poisonous seeds

The feline panleukopenia virus rescue strain FPV BJ05C was continuously passed on F81 cells for 35 generations (that is, on the basis of the virus strain FPV BJ05C strain for 30 generations), the TCID ₅₀ of each generation of the virus was determined, and the fifth and tenth were selected. , 15, 20, 25, 30, 35, and 40 generations of viruses were inactivated to prepare vaccines, and 5 healthy susceptible puppies were vaccinated, observed for 21 days and challenged. The results showed that the feline panleukopenia virus FPV BJ05C strain could maintain high virus content, good immunogenicity and immune protection after passage for at least 40 generations. After the 5th generation (including the 5th generation virus, that is, the virus strain with preservation number CGMCCNo.19988), the virus content of each generation is stable, not less than 10 ^6.0 TCID ₅₀ , and the HA titer is above 2 ⁹ , which has reached standard for vaccines. The results of the challenge test showed that after each generation of virus inactivated inoculated susceptible kittens, the immunized animals could resist the virulent challenge, and the challenge kittens survived, while the control group had different degrees of morbidity or death. According to the above results, in order to ensure the good immunogenicity and safety of the virus seed, the present invention determines that the maximum number of passages of the virus is 30 generations, the original virus seed is 1 to 5 generations, and the basic virus seed is the 6th to 15th generations. The maximum number of passages of the virus seeds for production is limited to 3 generations (16th to 18th generations).

1.2.8 Storage period of poisonous seeds

The original seeds, basic seed batches, and working seed batches of different generations of feline panleukopenia virus FPV BJ05C strain were compared under different conditions and tested for value-added stability. The results showed that the virus content was ≥10 ^6.0 TCID ₅₀ /ml when stored at -70°C, freeze-dried virus seeds for 30 months, and wet virus seeds for 18 months; when stored at -20°C, freeze-dried virus seeds Stored for 30 months, and the wet poison virus seeds were stored for 12 months, and the virus content was all ≥10 ^6.0 TCID ₅₀ /ml. The virus seeds within the storage period were successively passaged for 3 times, and there was no significant difference in the virus content, and the virus proliferation was stable. Based on the above results, in order to ensure the quality of vaccine production, the freeze-dried feline panleukopenia virus seed was stored at -20°C for about 30 months, and at -70°C for about 30 months; the wet virus seed was stored in -20 ℃ storage period is about 12 months, storage below -70 ℃, the storage period is about 18 months.

2. Production cells

2.1 Cell source

F81 cells were purchased from China (Wuhan) Type Culture Collection.

2.2 Selection of cells

In the culture process of the virus, the present invention selects the passage cell line F81 for virus propagation. As a result, the virus is stable on the F81 cell and has a high proliferation titer. The feline panleukopenia virus FPV BJ05C strain is inoculated with the F81 cell for 72 hours. The pathological condition is as follows: Figure 1.

The virus particles of the strain FPV BJ05 were observed under a transmission electron microscope, as shown in Figure 2. The results of FPV indirect immunofluorescence test are shown in Figure 3.

2.3 Establishment of cell lines

F81 cells were selected as the cell line for seedling production. The passage range is controlled at 40 to 55 passages, and the invention establishes an original cell bank, a basic cell bank and a working cell bank. Among them, the original cell bank: the purchased F81 cells at passage 0 were continuously passaged for 5 generations, 1ml/vial, for a total of 30 cells; the basic cell bank: the frozen original cell seeds were recovered, and the cells were serially passaged 10 times to prepare the basic cell seeds. . 1ml/tube, 120 tubes in total; working cell bank: The recovered basal cells are serially passaged 10 times to prepare working cell seeds, 1ml/tube, 250 tubes in total. The cells of each generation are tested according to the appendix of "Chinese Veterinary Pharmacopoeia", and there should be no contamination of bacteria, molds, mycoplasma and foreign viruses.

2.4 Identification of cell lines

According to the current "Chinese Veterinary Pharmacopoeia" appendix on the "production and inspection of cell standards" regulations, all meet the standards.

Example 2 Preparation of feline panleukopenia virus inactivated vaccine

The preparation process of feline panleukopenia virus inactivated vaccine (FPV BJ05C strain) is as follows. Judging from the quality inspection results of 3 batches of vaccines prepared in the laboratory and 5 batches of vaccines produced in the intermediate trial, the quality of the vaccine products prepared by this process is stable, and the efficacy inspection results fully meet the established quality standards.

1. Preparation of virus solution

In the process of virus reproduction, many factors will affect the proliferation of cells and viruses. The replication of FPV requires the DNA polymerase of the host cell. Cells with strong proliferation ability and in the mitotic stage are most suitable for FPV proliferation. Therefore, when FPV is propagated, it is used to inoculate and culture at the same time as the cells; the serum concentration in the culture medium significantly affects the replication of the virus. After a series of comparative tests, the serum concentration of the cell culture solution was 10%, and the serum concentration of the maintenance solution was 2%, which was able to reproduce a stable, high-titer virus solution; The dosage and collection time were compared, and the results showed that the pH value of the cell maintenance solution was controlled between 7.2 and 7.4 during virus propagation, and the cell concentration was selected from 1×10 ⁵ to 5×10 ⁵ cells/ml when the virus was inoculated. The seed virus with the poison dose of 1% (v/v) and the virus content of 10 ^6.0 TCID ₅₀ /ml or more and the best harvest time of the virus were 72-96 hours, and the virus liquid with high virus content could be obtained.

In this example, the serum concentration in the cell culture fluid (trade name 1640, purchased from Gibco Company) is 10%, pH value is 7.3, and the maintenance fluid (trade name MEM, purchased from Beijing Huabaitai Biotechnology Co., Ltd.) The concentration of medium serum is 2%, the concentration of F81 cells is 3×10 ⁵ cells/ml when inoculated, and cat panleukocytes with a virus content of 10 ^6.0 TCID ₅₀ /ml are inoculated according to the cell inoculation dose of 1% (v/v) Reduce the virus FPV BJ05C strain, the virus harvest time is 72h.

2. Inactivation process

Formaldehyde solution with final concentration of 0.1%, 0.2%, 0.3%, 0.4% and β-propiolactone with final concentration of 0.05%, 0.03%, 0.025%, 0.02% were selected to carry out inactivation comparison test of FPV BJ05C, and the test results The results showed that 0.025% β-propiolactone could completely inactivate feline panleukopenia virus after being treated at 4°C for 48h. The results showed that β-propiolactone had little damage to the antigen, and it was hydrolyzed at 37°C for 2h after inactivation. , β-propiolactone toxicity disappeared. FPV antigen is added with 0.3% formaldehyde solution, which can be inactivated at 37℃ for 48 hours to completely inactivate the virus.

3. Emulsification process

Feline Panleukopenia Virus Inactivated Vaccine is a commercialized aluminum hydroxide adjuvant from Thermo Fisher Scientific at a final concentration of 1000 μg/ml and a final concentration of 0.025% β-propiolactone inactivated feline panleukopenia virus. (FPV BJ05C strain) was obtained by emulsification. Before adjuvant use, shake the aluminum adjuvant bottle gently. Before emulsification, it will be thawed and mixed with the inactivated virus solution of the same batch, and the aluminum hydroxide adjuvant will be dropped into the virus solution under aseptic conditions until the final volume ratio of adjuvant and virus solution is 1:3, and a homogenizer will be used at 200 r. /min stir evenly; in order to stabilize the interface and eliminate air bubbles, to obtain the best emulsification effect, it needs to stand for 30min and then dispense.

4. Inspection quality standards for semi-finished products

4.1 Sterility test

According to the current appendix of "Chinese Veterinary Pharmacopoeia", it should be aseptically grown.

4.2 Determination of virus content

The virus seeds were serially diluted 10 times with DMEM medium, 100 μl of each dilution was added to 96-well cell culture plate, and 8 repetitions were made for each dilution, and then F81 cell suspension was added, 100 μl per well (cell density) 2 × 10 ⁵ /mL, DMEM containing 10% newborn calf serum), and normal cell culture was set as a control, placed in a 37°C, 5% CO ₂ incubator, and replaced with 2% newborn calf serum after 12 h Continue to observe for 4 to 5 days under the same culture conditions, and observe cytopathic changes (CPE) day by day. Cells pyknosis, aggregate, increase granules, and some cells fuse, fall off, and have many voids, which are judged as CPE. The number of wells with cytopathic changes was recorded, and the TCID ₅₀ was calculated according to the Reed-Muench method. The results showed that the viral liquid was not less than 10 ^6.0 TCID ₅₀ per milliliter.

4.3 Inactivation test

Take the inactivated virus solution, dilute it according to 10 ⁰ , 10 ^-1 , and 10 ^-2 , and inoculate it in 3 flasks of F81 cells at 2% (the cell density is 3×10 ⁵ /ml) at 37°C, 5% CO ₂ was cultured for observation, and a virus control was set at the same time, and the cells were continuously passaged for 3 generations. The results showed no cytopathic changes. The three batches of semi-finished products produced in the laboratory are all qualified.

5. Finished product inspection quality standard

5.1 Safety inspection standards

In order to ensure the safety of the vaccine, the present invention has successively conducted a single-dose, repeated single-dose, and one-overdose safety test on healthy kittens, adult cats, and pregnant cats for the five batches of vaccines prepared. The test results show that there is no adverse local reactions such as infarction, redness and swelling in the injection site of a single dose, single dose repeated, and one overdose inoculation of kittens, adult cats, and pregnant cats. There is no pain when pressing, no depression, loss of appetite or abolition The total number of leukocytes before and after inoculation of kittens and adult cats was not significantly different; the weight gain of kittens was basically the same as that of the control group, and there were no pathological changes in all organs at autopsy; pregnancy and delivery of pregnant cats were normal , No miscarriage, premature birth, stillbirth and mummified fetus. The results showed that the vaccine was safe for kittens, adult cats and pregnant cats with a single dose, repeated single dose and one overdose.

5.2 The formulation of efficacy test standards

5.2.1 Correlation test between antibody titer and immune challenge protection

One batch of feline panleukopenia virus inactivated vaccine (batch number 201801) that passed the safety test was immunized with different doses of 0.5ml/piece, 1ml/piece, and 2ml/piece respectively. Infected cats (FPV neutralization titer not higher than 1:4 or HI antibody not higher than 1:8), each of 5 cats, 21 days after immunization, together with the control group experimental animals, blood was collected, and the corresponding antibody titer in the serum was determined. 10 ^6.0 TCID ₅₀ /ml FPV BJ05 strain 5th generation virulent was administered orally with 2ml for 21 consecutive days. The results showed that all cats with serum FPV HI<1:32 became ill, and cats with FPV HI>1:32 were resistant to FPVBJ05 strain virulent challenge. According to the test results of the correlation between vaccine immunization antibody titer and challenge protection, the HI antibody titer that can protect cats against FPV virulent challenge is not less than 1:32.

5.2.2 Minimum immunization dose

Three batches of feline panleukopenia virus inactivated vaccines 201801, 201802 and 201803 that passed the safety test were immunized with different doses of 0.5ml/piece, 1ml/piece, 1.5ml/piece, and 2ml/head respectively for 45 10 healthy sensitive kittens (FPV HI antibody not higher than 1:8) at the age of 60 days, and another 10 were not vaccinated as controls. FPV hemagglutination inhibition titer determination. On the 21st day after inoculation, the fifth generation culture of FPV BJ05 strain was administered orally for challenge, and the test cats were continuously observed for 21 days to record the protection status of the test cats. According to the detection of serum antibodies and the protective effect of challenge immunity, the minimum immune dose of the inactivated vaccine was determined to be 1.0ml/piece, and the dose to be used was determined to be 1.0ml/piece.

5.2.3 Correlation between mouse serological potency test and cat serological potency test

Three batches of feline panleukopenia virus inactivated vaccines 201801, 201802 and 201803 produced in the laboratory were subcutaneously immunized at doses of 0.05ml/vesicle, 0.10ml/vessel, 0.15ml/vessel, and 0.20ml/vessel for 19-21 days of age BALB/c mice and 5 Chinese pastoral cats aged 45-60 days, respectively, on the 7th, 14th and 21st days after immunization, together with the control group test mice and cats, the HI antibody titers were determined, and the mice were analyzed. Correlation with HI antibodies in cats. The results showed that when mice were immunized with a dose of 0.1 ml/mouse, the FPV HI titer was not lower than 1:32 after 14 days of inoculation, which could achieve the same immune efficacy as immunizing cats with a dose of 1.0 ml/mouse. Therefore, when formulating product quality standards, it is determined that the method of detecting FPV HI titer in mouse serum can be used to test the efficacy of the vaccine, that is, 14 days after the vaccine is immunized with 0.1ml/dose of mice, FPV hemagglutination inhibitory antibody If the titer is not less than 1:32, it is qualified.

6. Immune generation phase and immune persistence phase test

Three batches of feline panleukopenia virus inactivated vaccines 201801, 201802 and 201803 that passed the safety test were immunized to healthy susceptible cats respectively. In order to further grasp the immunization efficacy and immunization duration, formulate a reasonable immunization program to ensure that the immunized cats maintain a high and lasting antibody level, and conduct antibody detection on the immunized cats at different times to grasp the law of the growth and decline of their antibodies.

The test results showed that the FPV hemagglutination inhibitory titer in serum increased continuously with time, that is, the immune protection against FPV was generated 14 days after immunization, reached a peak at 2 months, and then slowly decreased to below the protective level. . Based on the above experimental results, the immune protection period of the vaccine is set as 6 months. The results of the protection period without challenge test show that the protection period of the vaccine is at least 6 months. Therefore, the vaccine is vaccinated every 6 months to ensure that Immune potency.

7. Determination of maternal antibody level and immunization period

By studying the changes of protective antibodies in serum of immunized kittens at different times after weaning, the influence of maternal antibodies on vaccine immunization was determined, so as to select an appropriate vaccine immunization time. The results showed that the maternal antibody level of kittens was still high at 1 week after weaning, which seriously affected the immune effect of the vaccine; 2 to 3 weeks after weaning, the maternal antibody level of kittens had been reduced to a low level, and the immunity to the vaccine was significantly reduced. The impact is small, and the immunized kittens can establish better active immunity. Therefore, according to the change rules of maternal antibodies and the changes of protective antibodies in the serum of immunized cats at different immunization times, the best time for vaccine immunization is 14 to 21 days after weaning.

8. Determination of immunization frequency

This vaccine is used to prevent panleukopenia in cats, and the immunization period is 6 months. 1ml/only, subcutaneous injection. According to the duration of maternal antibody of kittens, kittens are immunized once at 14 to 21 days of age after weaning, boosted once after 2 to 3 weeks, and then vaccinated once every 4 weeks, 3 times in a row, 1 per time , and then vaccinate every 6 months.

9. Shelf life of vaccines

The present invention conducts experimental research on the storage period of three batches of inactivated vaccines (201801, 201802, 201803) prepared, and the three batches of vaccines are stored at 2 to 8°C for 9, 12, 15, and 18 months. Samples were taken to test their characters, safety and immune efficacy. The results showed that the three batches of vaccines were stored at 2 to 8 °C for 18 months without significant changes in traits, and the sterility inspection and safety met the requirements of quality standards. In the efficacy test, the HI antibody titer of one mouse was 1:32 after immunizing mice with 1801 batches of vaccines stored for 18 months. The HI antibody titer of one cat after 18 months of immunizing cats with samples of 201801 and 201802 vaccines was 1:32 1:32, but the average HI antibody level of the immunized cat is 1:64. Considering the loss of titer caused by the vaccine during transportation and use, the present invention sets the shelf life of the vaccine as 15 months at 2-8°C. .

10. Comparison of immune efficacy with domestic similar commercial vaccines

10.1 Safety comparison test

10 healthy susceptible cats aged 45-60 days (FPV HI antibody titer not higher than 1:8) were randomly divided into two groups A and B, 5 in each group. Group A was subcutaneously injected with the FPV BJ05C inactivated vaccine developed , 1 head (1ml) / piece, group B subcutaneously injected "Miaosanduo" (feline rhinotracheitis, inflamed cup virus disease, panleukopenia triple inactivated vaccine produced by Zoetis, production batch number: 1620390A) 1 portion (1ml)/cat, and another 5 healthy susceptible cats were selected not to be vaccinated as negative controls. The local and systemic (spirit, appetite, body temperature and feces, etc.) clinical manifestations of the vaccinated cats were observed for 21 consecutive days to compare the safety of the vaccine. The results showed that within 21 days of the observation period, all the observation items of the vaccinated cats were normal, the two vaccines were safe for cats, and there was no difference in safety between the two.

10.2 Comparative test of immune effect

The FPV BJ05C inactivated vaccine of the present invention and "Miaosanduo" were subcutaneously inoculated into 45-day-old healthy and susceptible Chinese pastoral cats (FPV HI antibody not higher than 1:8) for each 5, 1ml/head portion/cat , and another 5 45-day-old healthy and susceptible Chinese pastoral cats were not vaccinated as negative controls. Before immunization and 7 days, 14 days, 21 days, 30 days, 2 months, April, and 6 months after immunization, blood was collected to separate serum, and FPV hemagglutination inhibitory titer (HI) in serum of immunized cats was detected. The results showed that about 2 months after immunization with the two vaccines, the FPV hemagglutination inhibitory titers in the serum of immunized cats all reached the peak, and remained at a high level 6 months after immunization. And during the 6-month detection period, there was no significant difference in FPV HI antibody levels between the two groups. However, the strains used in the present invention are strains isolated after a large number of epidemiological investigations in the laboratory, and the inactivated vaccine is more compatible with domestic circulating strains, which is beneficial to the prevention and control of feline panleukopenia.

Table 1 shows the change situation of serum HI titer by immunizing cats with feline panleukopenia virus inactivated vaccine of the present invention.

Table 1 Changes in serum HI titer of immunized cats

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

sequence listing

<110> Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences

<120> Feline panleukopenia virus FPV BJ05 strain and its application

<130> KHP201110477.3

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5118

<212> DNA

<213> Feline panleukopenia virus

<400> 1

ctttagaacc aactgaccaa gttcacgtac gtatgacgtg atgacgcgcg ctgcgcgcgc 60

tgcctacggc agtcacacgt catacgtacg ctccttgatc agttggttct aaagaatgat 120

aggcggtttg tgtgtttaaa cttgggcggg aaaaggtggc gggctaattg tgggcgtggt 180

taaaggtata aaagacaaac catagaccgt tactgacatt cgcttcttgt ctttgacaga 240

gtgaacctct cttactttga ctaaccatgt ctggcaacca gtatactgag gaagttatgg 300

agggagtaaa ttggttaaag aaacatgcag aaaatgaagc attttcgttt gtttttaaat 360

gtgacaacgt ccaactaaat ggaaaggatg ttcactggaa caactatacc aaaccaattc 420

aaaatgaaga gctaacatct ttaattagag gagcacaaac agcaatggat caaaccgaag 480

aagaagaaat ggactgggaa tcggaagttg atagtctcgc caaaaagcaa gtacaaacct 540

ttgatgcatt aattaaaaaa tgtctttttg aagtctttgt ttctaaaaat atagaaccaa 600

atgaatgtgt ttggtttatt caacatgaat ggggaaaaga tcaaggctgg cattgtcatg 660

ttttacttca tagtaagaac ttacaacaag caactggtaa atggctacgc agacaaatga 720

atatgtattg gagtagatgg ttggtgactc tttgttcggt aaacttaaca ccaactgaaa 780

agattaagct cagagaaatt gcagaagata gtgaatgggt gactatatta acatacagac 840

ataagcaaac aaaaaaagac tatgttaaaa tggttcattt tggaaatatg atagcatatt 900

actttttaac aaagaaaaaa attgtccaca tgacaaaaga aagtggctat tttttaagta 960

ctgattctgg ttggaaattt aactttatga agtatcaaga cagacatact gtcagcacac 1020

tttacacgtt ctaactcctc tgactccgga cgtagtggac cttgcactgg aaccgtggag 1080

tactccagat acgcctattg cagaaactgc aaatcaacaa tcaaaccaac ttggcgttac 1140

tcacaaagac gtgcaagcga gtccgacatg gtccgaaata gaggcagacc taagagccat 1200

ctttacttct gaacaattgg aagaagattt tcaagacgac ttggattaag gtacgatggc 1260

acctccggca aagagagcca ggagaggtaa gggtgtgtta gtaaagtggg gggagggaa 1320

aaatttaata acttaactaa gtatgtgttt ttttatagga cttgtgcctc caggttataa 1380

atatcttggg cctgggaaca gtcttgacca aggagaacca actaaccctt ttgacgccgc 1440

tgcaaaagaa cacgacgaag cttacgctgc ttattttcgc tctggtaaaa acccatactt 1500

atatttttcg ccagcagatc aacgctttat agatcaaaat aaggacgcta cagattgggg 1560

ggggaaaata ggacattatt tttttagagc taaaaaagca attgctccag tattaactga 1620

tacaccagat catccatcaa catcaagacc aacaaaacca actaaaagaa gtaaaccacc 1680

acctcatatt ttcatcaatc ttgcaaaaaa aaaaaaagcc ggtgcaggac aagtaaaaag 1740

agacaatctt gcaccaatga gtgatggagc agttcaacca gacggtggtc aacctgctgt 1800

cagaaatgaa agagctacag gatctgggaa cgggtctgga ggcgggggtg gtggtggttc 1860

tgggggtgtg gggatttcta cgggtacttt caataatcag acggaattta aatttttgga 1920

aaacggatgg gtggaaatca cagcaaactc aagcagactt gtacatttaa atatgccaga 1980

aagtgaaaat tataaaagag tagttgtaaa taatatggat aaaactgcag ttaaaggaaa 2040

catggctaaa aacaggaatt aactatacta atatatttaa tacttatggt cctttaactg 2100

cattaaataa tgtaccacca gtttatccaa atggtcaaat ttgggataaa gaatttgata 2160

ctgacttaaa accaagactt catgtaaatg caccatttgt ttgtcaaaat aattgtcctg 2220

gtcaattatt tgtaaaagtt gcgcctaatt taacaaatga atatgatcct gatgcatctg 2280

ctaatatgtc aagaattgta acttactcag atttttggtg gaaaggtaaa ttagtattta 2340

aagctaaact aagagcatct catacttgga atccaattca acaaatgagt attaatgtag 2400

ataaccaatt taactatcta ccaaataata ttggagctat gaaaattgta tatgaaaaat 2460

ctcaactagc acctagaaaa ttatattaat atacttacta tgtttttatg tttattacat 2520

atcaactagc acctagaaaa ttatattaat atacttacta tgtttttatg tttattacat 2580

attattttaa gattaattaa attacaacat agaaatattg tacttgtatt tgatatagga 2640

tttagaaggt ttgttatatg gtatacaata actgtaagaa atagaagaac atttagatca 2700

tggttagtag tttgttttat aaaatgtaat tgtaaactat taatgtatgt tgttatggtg 2760

tgggtggttg gttggtttgc ccttagaata tgttaaggac caaaaaaatc aataaaagac 2820

atttaaaact taatggtctc gtatactgtc tataaggtga actaacctta ccataagtat 2880

caatctgtct ttaagggggg ggtgggtggg agatgcacaa tatcagtaga ctgactggcc 2940

tggttggttg ctctgcttaa tcaaccagac cgcgtagcgg tctggttgat taagcgcaac 3000

caaccaggcc agtcagtcta ctgatattgt gcatctccca cccacccccc ccttaaagac 3060

agattgatac ttaaaacagg aattaactat actaatatat ttaatactta tggtccttta 3120

actgcattaa ataatgtacc accagtttat ccaaatggtc aaatttggga taaagaattt 3180

gatactgact taaaaccaag acttcatgta aatgcaccat ttgtttgtca aaataattgt 3240

cctggtcaat tatttgtaaa agttgcgcct aatttaacaa atgaatatga tcctgatgca 3300

tctgctaata tgtcaagaat tgtaacttac tcagatttttt ggtggaaagg taaattagta 3360

tttaaagcta aactaagagc atctcatact tggaatccaa ttcaacaaat gagtattaat 3420

gtagataacc aatttaacta tctaccaaat aatattggag ctatgaaaat tgtatatgaa 3480

aaatctcaac tagcacctag aaaattatat taatatactt actatgtttt tatgtttatt 3540

acatatcaac tagcacctag aaaattatat taatatactt actatgtttt tatgtttatt 3600

acatattatt ttaagattaa ttaaattaca acatagaaat attgtacttg tatttgatat 3660

aggatttaga aggtttgtta tatggtatac aataactgta agaaatagaa gaacatttag 3720

atcatggtta gtagtttgtt ttataaaatg taattgtaaa ctattaatgt atgttgttat 3780

ggtgtgggtg gttggttggt ttgcccttag aatatgttaa ggaccaaaaa aatcaataaa 3840

agacatttaa aacttaatgg tctcgtatac tgtctataag gtgaactaac cttaccataa 3900

gtatcaatct gtctttaagg ggggggtggg tgggagatgc acaatatcag tagactgact 3960

ggcctggttg gttgctctgc ttaatcaacc agaccgcgta gcggtctggt tgattaagcg 4020

caaccaacca ggccagtcag tctactgata ttgtgcatct cccacccacc ccccccttaa 4080

agacagattg atacttaaaa caggaattaa ctatactaat atatttaata cttatggtcc 4140

tttaactgca ttaaataatg taccaccagt ttatccaaat ggtcaaattt gggataaaga 4200

atttgatact gacttaaaac caagacttca tgtaaatgca ccatttgttt gtcaaaataa 4260

ttgtcctggt caattatttg taaaagttgc gcctaattta acaaatgaat atgatcctga 4320

tgcatctgct aatatgtcaa gaattgtaac ttactcagat ttttggtgga aaggtaaatt 4380

agtatttaaa gctaaactaa gagcatctca tacttggaat ccaattcaac aaatgagtat 4440

taatgtagat aaccaattta actatctacc aaataatatt ggagctatga aaattgtata 4500

tgaaaaatct caactagcac ctagaaaatt atattaatat acttactatg ttttttatgtt 4560

tattacatat caactagcac ctagaaaatt atattaatat acttactatg ttttttatgtt 4620

tattacatat tattttaaga ttaattaaat tacaacatag aaatattgta cttgtatttg 4680

atataggatt tagaaggttt gttatatggt atacaataac tgtaagaaat agaagaacat 4740

ttagatcatg gttagtagtt tgttttataa aatgtaattg taaactatta atgtatgttg 4800

ttatggtgtg ggtggttggt tggtttgccc ttagaatatg ttaaggacca aaaaaatcaa 4860

taaaagacat ttaaaactta atggtctcgt atactgtcta taaggtgaac taaccttacc 4920

ataagtatca atctgtcttt aagggggggg tgggtgggag atgcacaata tcagtagact 4980

gactggcctg gttggttgct ctgcttaatc aaccagaccg cgtagcggtc tggttgatta 5040

agcgcaacca accaggccag tcagtctact gatattgtgc atctcccacc cacccccccc 5100

ttaaagacag attgatac 5118

Claims (10)

1. The feline panleukopenia virus FPV BJ05 strain is characterized in that the preservation number is CGMCC No. 19988.

2. The use of the rescued strain of FPV BJ05 strain of claim 1 for any of the following:

1) for the preparation of vaccines;

2) is used for preparing diagnostic reagent for feline panleukopenia virus infection and relevant diseases caused by the infection.

3. A composition comprising a rescued strain of the FPV BJ05 strain of claim 1 after inactivation and a pharmaceutically acceptable carrier.

4. An immunogenic composition comprising the composition of claim 3.

5. A vaccine composition comprising the immunogenic composition of claim 4.

6. The preparation method of the inactivated vaccine for the feline panleukopenia virus is characterized by comprising the following steps:

(1) infectious cloning the feline panleukopenia virus FPV BJ05 strain of claim 1, inoculating the obtained rescued strain to susceptible cells for culture to obtain virus solution;

(2) inactivating the harvested virus liquid with beta-propiolactone;

(3) and mixing the inactivated virus solution with an adjuvant to obtain the virus-free vaccine.

7. The method of claim 6, wherein the susceptible cells of step (1) are F81 cells; and/or

The adjuvant in the step (3) is an aluminum adjuvant, preferably an aluminum hydroxide adjuvant.

8. The method of claim 6 or 7, wherein step (2) comprises: and (3) adding the harvested virus liquid into beta-propiolactone with the final concentration of 0.02-0.05% to inactivate for 36-48 h at 4-10 ℃, stirring once every 2h during the inactivation, and hydrolyzing the inactivated virus liquid at 37 ℃ for 1-2 h.

9. Use of a vaccine composition according to claim 5 or an inactivated feline panleukopenia virus vaccine produced by the process according to any one of claims 6 to 8 in the manufacture of a biological product for the treatment or prevention of infection by feline panleukopenia virus and related diseases caused by infection thereof.

10. The use of claim 9, wherein the disease is characterized by the onset of bilateral fever, vomiting, diarrhea, dehydration, marked leukopenia and hemorrhagic enteritis in the affected feline.

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