CN111876391A

CN111876391A - Feline panleukopenia virus FPV BJ05 strain and application thereof

Info

Publication number: CN111876391A
Application number: CN202010728830.1A
Authority: CN
Inventors: 梁瑞英; 梁琳; 崔尚金
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2020-07-23
Filing date: 2020-07-23
Publication date: 2020-11-03

Abstract

The invention provides a feline panleukopenia virus FPV BJ05 strain and application thereof. The strain FPV BJ05 has the advantages of good immunogenicity, good culture characteristics, stable titer and the like, the FPV BJ05 strain is subjected to infectious cloning to obtain a rescue strain FPV BJ05C with genetic markers, the rescue strain is inoculated to susceptible cells, a culture is harvested, and after being inactivated by beta-propiolactone, the inactivated vaccine is mixed and emulsified with an aluminum hydroxide adjuvant to prepare the inactivated vaccine for the feline panleukopenia virus. Experiments show that the inactivated vaccine for feline panleukopenia virus provided by the invention can prevent diseases caused by FPV for healthy kittens after weaning after immunization, and the vaccine has the advantages of safety, quick response, long immune period and the like.

Description

Feline panleukopenia virus FPV BJ05 strain and application thereof

Technical Field

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

Background

Feline panleukopenia virus is a single-stranded DNA virus without an envelope, a virus that causes high infectivity and lethality in cats and felines. Can cause leukopenia (FPD) in cats and felines. The virus is in a world epidemic trend and is considered to be a virus with the widest infection range and the strongest pathogenicity of the parvovirus of the carnivore. The virus was first discovered in 1928 and was formally named feline panleukopenia virus in 1935. FPV is transmitted via the fecal-oral route, primarily through contact with infected body fluids, feces, or other contaminants, as well as via fleas. FPV are environmentally robust and survive for at least one year in infected tissue samples. The contamination is a major source of transmission and the owner of the cat can transmit highly contagious viruses to cats indoors through shoes, clothing, etc. After the infection of the virus through nose or mouth feel for 18-24 h, the virus begins to replicate in the oropharynx, viremia appears after 2-7 days, and virus replication mainly occurs in mitotic tissues, so that the lymphatic tissues, bone marrow and intestinal mucosa of cats with the age of more than 6 weeks are most frequently affected.

Disclosure of Invention

The invention aims to provide a feline panleukopenia virus FPV BJ05 strain with good immunogenicity, good culture characteristics and stable titer.

Another objective of the invention is to provide an application of a rescued strain of feline panleukopenia virus FPV BJ05 strain (FPVBJ05C strain) in preparing vaccines.

To achieve the object of the present invention, in a first aspect, the present invention provides feline panleukopenia virus (felineplanukopenia virus) FPV BJ05 strain. The inventor isolated a cat leukopenia virus from a certain animal hospital in Beijing in 2017, and isolated the virus from diseased cat feces with vomit, diarrhea and severe hemorrhagic enteritis. The domesticated strain is named as FPV BJ05 strain, and compared with other separated strains, the domesticated strain has high cell proliferation titer and good immunogenicity to cats. A rescued strain of FPV BJ05 strain, FPV BJ05C strain (vaccine strain), was obtained by infectious cloning. Compared with a wild strain (FPV BJ05 strain), the genetic marker is added through site-directed mutation, namely the genome 2980-2985bp of the FPV BJ05 strain is mutated from CTCAAG to CTCGAG, the rescued strain and the wild strain are proved to have the same biological characteristics, and are used as a vaccine virus after being passaged by a cat kidney cell line (F81), and the virulent strain for titer detection is the wild strain FPV BJ05 at 7-10 generations. The strain can cause more than 90% of infection of F81 cells. The genome sequence of the FPV BJ05 strain is shown as SEQ ID NO. 1.

The Feline panleukopenia virus (Feline panleukopenia virus) FPV BJ05 strain is currently deposited in the common microorganism center of China Committee for culture Collection of microorganisms, No. 3 of Xilu 1 of Beijing Korean district, No. 3 of the institute of microbiology of the Chinese academy of sciences, zip code 100101, preservation No. CGMCC No.19988, and the preservation date is 2020, 5 months and 28 days.

In a second aspect, the invention provides the use of a rescued strain of feline panleukopenia virus FPV BJ05 strain (FPVBJ05C strain) as described in any one 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.

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 immunogenic composition described above.

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

(1) carrying out infectious cloning on the feline panleukopenia virus FPV BJ05 strain, inoculating the obtained rescue strain to susceptible cells for culture, and obtaining virus solution;

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

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

In the foregoing method, step (1) includes: the method comprises the steps of analyzing Y-shaped structures and U-shaped structures at the 3 'end and the 5' end of a virus by bioinformatics, amplifying genome sequence (SEQ ID NO:1) of feline panleukopenia virus FPV BJ05 strain, inserting pBluescript II (+) vector, constructing a reverse genetics platform, transfecting F81 cells, carrying out virus rescue, obtaining a rescued strain, inoculating the rescued strain to susceptible cells (such as F81 cells) for culture, and obtaining virus liquid.

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

In the foregoing method, step (2) includes: inactivating the harvested virus liquid by adopting beta-propiolactone with the final concentration of 0.02-0.05% at the temperature of 4-10 ℃ for 36-48 h, stirring once every 2h during the inactivation, and hydrolyzing the inactivated virus liquid at the temperature of 37 ℃ for 1-2 h. Preferably, the harvested virus liquid is inactivated by beta-propiolactone with a final concentration of 0.03% at 4 ℃ for 48h, and is stirred once every 2h, and the inactivated virus liquid is hydrolyzed at 37 ℃ for 2 h. And (4) preserving the inactivated virus liquid at 2-8 ℃.

Preferably, the inactivated feline panleukopenia virus vaccine is prepared by emulsifying an aluminum hydroxide adjuvant with a final concentration of 1000 mug/ml and beta-propiolactone inactivated feline panleukopenia virus (FPV BJ05C strain) with a final concentration of 0.03%.

The emulsification process adopts commercial aluminum hydroxide adjuvant of American Saimerfic company as the emulsification process, and the adjuvant can be directly used for emulsification preparation of vaccines after degerming; before adjuvant use, the aluminum adjuvant bottle was gently shaken. Before emulsification, the inactivated virus liquid and the same batch of virus liquid are unfrozen and mixed, an aluminum hydroxide adjuvant is dropped into the virus liquid under the aseptic condition, the volume ratio of the adjuvant to the virus liquid is 1:3, and a homogenizer is used for stirring uniformly at 200 r/min. In order to stabilize the interface and eliminate air bubbles, and obtain the best emulsification effect, the emulsion needs to be subpackaged after standing for about 30 minutes.

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

The disease is characterized by the onset of bilateral fever, vomiting, diarrhea, dehydration, significant leukopenia, hemorrhagic enteritis, etc. in the affected feline.

The strain saved FPV BJ05C strain of the feline panleukopenia virus FPV BJ05 strain provided by the invention has the advantages of good immunogenicity, good culture characteristics, stable titer and the like, the genetic marker is added into the saved strain, the wild strain and the vaccine strain are convenient to distinguish, the FPV BJ05C strain is inoculated with susceptible cells, the culture is harvested, and the inactivated beta-propiolactone is mixed and emulsified with an aluminum hydroxide adjuvant to prepare the inactivated vaccine of the feline panleukopenia virus. Experiments show that the inactivated vaccine for feline panleukopenia virus provided by the invention can prevent FPD (focal plane partial volume) diseases of healthy kittens after weaning by immunizing the weaned cats with the inactivated vaccine for feline panleukopenia virus, and the vaccine has the advantages of safety, quick response, lasting immune period and the like.

Drawings

FIG. 1 is a diagram showing cytopathic effects of F81 cells inoculated with FPV BJ05C strain of feline panleukopenia virus in a preferred embodiment of the present invention. Wherein, A: cytopathic effect after 72h of inoculation; b: normal cells served as negative controls.

FIG. 2 shows the virions of strain FPV BJ05C observed under a transmission electron microscope in a preferred embodiment of the invention.

FIG. 3 shows the results of indirect immunofluorescence assay of feline panleukopenia virus strain FPV BJ05C in accordance with a preferred embodiment of the present invention. Wherein, A: fluorescence detection results of the FPV BJ05C strain after being cultured on F81 cells for 24 hours; b: fluorescence detection of normal control cells.

Detailed Description

The invention provides a feline panleukopenia virus FPV BJ05 strain and application thereof. The inventor separates a porcine parvovirus from a certain animal hospital in Beijing in 2017, identifies the porcine parvovirus as a virulent strain, and names the domesticated strain as FPV BJ05 strain. The FPV BJ05 strain is used for carrying out virus rescue through reverse genetic operation, a rescue strain FPV BJ05C is obtained, the research and development work of inactivated vaccines of cat leukopenia viruses is developed, the FPV BJ05C strain of the cat leukopenia viruses with good immunogenicity and stable titer is rescued, and F81 cells which are most suitable for virus proliferation and inactivators, adjuvants and other conditions which accord with vaccine manufacture are selected. The production process, safety, protection rate, immunization program, minimum dosage, antibody duration and storage period of the biological product are tested, and a large amount of test data are obtained to prove that the vaccine is safe and effective. On the basis, pilot production is carried out, sampling inspection is carried out, the pilot product meets the established quality standard, safety test and efficacy test are carried out on the pilot product, the result proves that the vaccine can effectively prevent the feline panleukopenia caused by the feline panleukopenia virus, the inactivated vaccine of the feline panleukopenia virus (FPV BJ05C strain) is developed on the basis of laboratory test and intermediate trial production, and the result shows that the vaccine can effectively prevent the feline panleukopenia caused by the feline panleukopenia virus, and further verifies each quality standard of the vaccine.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the 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 commercially available products.

The cat species used in the following examples was a chinese garden cat.

Example 1 culture identification of feline panleukopenia Virus FPV BJ05C Strain

1. Source and standard of virus seed

According to the requirements of new biological product declaration and in combination with a large amount of test data obtained by the invention, the virus seeds for vaccine preparation are identified by referring to the Chinese veterinary pharmacopoeia (2005 edition).

1.1 sources of virulent seeds

The virus seed for preparing the biological product is an FPV BJ05C strain rescued by an infectious clone platform through cat leukopenia virus FPV BJ05, the FPV BJ05 strain is obtained by separating and domesticating cat feces from diseased cats in 2017, and the strain is separated from cat feces infected by typical cat leukopenia virus, has high proliferation titer on cells compared with other separated strains and good immunogenicity on cats, so the strain is selected for infectious clone to construct a reverse genetic operation platform, F81 cells are transfected to rescue the strain, a virus seed for vaccine production (FPV BJ05C) is obtained, and the virus seed is biologically identified. The strong toxicity for efficacy test is 7 th to 10 th generations of FPV BJ05 strain. The virus strain was passage 7 after passage on F81 cells.

1.2 seed Standard of Virus

1.2.1 erythrocyte agglutination value

A96-well U-type microplate was pipetted into 25. mu.l PBS (0.01mol/L, pH 7.0-7.4) per well, and 25. mu.l antigen was added to each well of the first row, which was then diluted in multiple aliquots until 25. mu.l was discarded in well 11. Adding 25 μ l PBS into each well, adding 25 μ l 1% pig red blood cell suspension, mixing well with a micro-oscillator, and acting at room temperature for 1 hr or acting in a refrigerator at 4 deg.C for 2 hr. In the case of the determination result, the highest dilution factor of the antigen with 50% erythrocyte agglutination was used as the determination end point. The result shows that the agglutination valence of the virus seeds to the pig red blood cells is not less than 1: 256.

1.2.2 Virus content

The virus seeds were serially diluted 10-fold in DMEM medium, 100. mu.l of each dilution was added to a 96-well cell culture plate in 8 replicates, followed by 100. mu.l of F81 cell suspension per well (cell density 2X 10)⁵/mL DMEM containing 10% newborn calf serum) and normal cell culture as a control, at 37 ℃ with 5% CO₂Culturing in an incubator, replacing a maintenance solution of 2% newborn calf serum after 12h, continuously observing for 4-5 days under the same culture condition, observing cytopathic effect (CPE) day by day, and judging CPE if cells shrink, gather and increase particles, and some cells fuse, fall off and have more gaps. The number of cytopathic wells was recorded and TCID was calculated according to the Reed-Muench method₅₀. The results show that the virus solution per milliliter is not less than 10^6.0TCID₅₀。

1.2.3 virulence

F81 cells are inoculated by the feline panleukopenia virus FPV BJ05C, and the virus generation is the 7 th-10 th generation of the FPV BJ 05C; the virus content per ml should not be less than 10^6.0TCID₅₀(ii) a The program is that 5 cats with the age of 45-60 days are injected intramuscularly, 2ml are added per cat, 5 control cats are set at the same time, after inoculation, the cats are kept separately, and 21 days are observed day by day. The judgment standard is mental depression, inappetence or abstinence; diarrhea, mucus or blood in the stool; feces (1:5 dilution) have a hemagglutination titer of not less than 1:64, and hemagglutination performance is inhibited by feline panleukopenia virus positive sera.

1.2.4 immunogenicity

Synchronously inoculating the virus seeds to F81 cells for proliferation, harvesting virus liquid, inactivating with beta-propiolactone with the final concentration of 0.03%, adding aluminum hydroxide adjuvant to the final concentration of 800ug/ml, and mixing well. 10 healthy susceptible cats (FPV HI antibody titer not higher than 1:8) with the age of 45-60 days are randomly divided into 2 groups of 5 cats. One group was injected subcutaneously with 1ml of the prepared vaccine, and the other group was used as a control and was not vaccinated. Feeding was isolated under the same conditions. Collecting blood 21 days later, collecting serum, and performing HI titer determination; meanwhile, the virulent strain for the subcutaneous injection efficacy test of all cats is FPV BJ05 strain 7 th-10 th generation culture solution (10)^6.0TCID₅₀Ml)1m1, collecting anal swabs every day, and observing for 21 days continuously, 5 cats in the control group should have diseases, and the judgment standard is as follows: depression, loss of appetite or abominable appetite; diarrhea, mucus or blood in the stool; feces (1:5 dilution) have a hemagglutination titer of not less than 1:64, and hemagglutination performance is inhibited by feline panleukopenia virus positive sera. HI antibody titer is not higher than 1: 8; the immunized 5 cats should be at least 4 healthy, alive, with HI antibody titers not below 1: 32.

1.2.5 purity test

The test of bacteria, mould and mycoplasma is carried out on the 1 st to 5 th generation of the established original seed lot, the 6 th to 15 th generation of the basic seed lot and the 16 th to 18 th generation of the production seed lot of the feline panleukopenia virus FPV BJ05C strain, and the test of exogenous viruses is carried out on the 3 rd, 9 th and 17 th generation of virus seeds, and the result shows that the original seed lot, the basic seed lot and the production seed lot which are established by the invention are free from the pollution of bacteria, mould, mycoplasma and exogenous viruses, and the virus seeds are pure.

1.2.6 specificity

Diluting the virus seeds to 200TCID by DMEM cell culture solution₅₀0.1ml, mixing with the inactivated anti-feline panleukopenia virus specific serum in equal amount, incubating in 37 deg.C water bath for 1h, synchronously inoculating well-grown F81 cell 3 bottles (cell density is 2 × 10)⁵/ml), simultaneously setting virus control, normal cell control and negative serum control, placing at 37 deg.C, and containing 5% CO₂And (5) culturing for 5-7 days in an incubator. Both the neutralized virus group and the normal cell control group should be free of cytopathic effect (CPE); virus control group and negative bloodCPE should be present in both the clear control groups.

1.2.7 use of the virus seeds

The feline panleukopenia virus rescued strain FPV BJ05C was continuously passaged on F81 cells for 35 generations (i.e., continuously passaged on the basis of the virulent strain FPV BJ05C for 30 generations), and the TCID of each generation of virus was determined₅₀And respectively inactivating viruses of 5 th, 10 th, 15 th, 20 th, 25 th, 30 th, 35 th and 40 th generations to prepare vaccines, inoculating 5 healthy and susceptible puppies, observing for 21 days and performing challenge test. The result shows that the feline panleukopenia virus FPV BJ05C strain can maintain higher virus content, good immunogenicity and immune protection after at least 40 passages. After the 5 th generation (including the 5 th generation virus, namely the virus strain with the preservation number of CGMCC No. 19988), the virus content of each generation is stable and is not less than 10^6.0TCID₅₀And all the HA titers are 2⁹Above, the standard of vaccine is achieved. The result of the challenge test shows that after virus inactivation of each generation is inoculated to susceptible kittens, immune animals can resist strong virus attack, the challenge kittens survive, and the control group suffers from diseases or death to different degrees. According to the results, in order to ensure good immunogenicity and safety of the produced virus seeds, the invention determines that the highest passage frequency of the virus is 30 generations, the original virus seeds are 1-5 generations, the basic virus seeds are 6-15 generations, and the highest passage frequency of the virus seeds for production is limited within 3 generations (16 th-18 th generations).

1.2.8 poison seed storage period

Carrying out different condition comparison and proliferation stability detection on the original seeds, the basic seed batches and the working seed batches of the feline panleukopenia virus FPV BJ05C strain. The results show that the virus seeds are preserved at the temperature of-70 ℃, the freeze-dried virus seeds are preserved for 30 months, the wet virus seeds are preserved for 18 months, and the virus content is more than or equal to 10^6.0TCID₅₀Per ml; storing at-20 deg.C, freeze-drying virus seeds for 30 months, storing wet virus seeds for 12 months, and storing virus content more than or equal to 10^6.0TCID₅₀And/ml. The virus seeds in the preservation period are continuously passed for 3 times, the virus content has no obvious difference, and the virus proliferation is stable. Based on the above results, in order to ensure the quality of vaccine production, feline panleukopenia virus was lyophilizedThe seed has a shelf life of about 30 months at-20 deg.C and about 30 months at-70 deg.C; the retention period of the seed of the damp poison at minus 20 ℃ is about 12 months, the seed of the damp poison is preserved at minus 70 ℃, and the retention period is about 18 months.

2. Production cell

2.1 cell origin

F81 cells were purchased from the Chinese (Wuhan) type culture Collection.

2.2 selection of cells

In the process of culturing the virus, the continuous cell line F81 is selected for virus propagation, so that the virus is stable on F81 cells and has high propagation titer, and the disease change condition is shown in figure 1 after the F81 cells are inoculated with the feline panleukopenia virus FPV BJ05C strain for 72 hours.

The virions of strain FPV BJ05 were observed under transmission electron microscopy as shown in FIG. 2. The results of the FPV indirect immunofluorescence assay are shown in FIG. 3.

2.3 establishment of cell lines

F81 cells were selected as the cell line for producing the vaccine. The passage range is controlled to be 40-55 generations, and the invention establishes an original cell bank, a basic cell bank and a working cell bank. Wherein the primary cell bank: continuously passaging the purchased F81 cells with 0 generation for 5 generations, wherein each cell is 1ml and 30 cells in total; basal cell bank: and (4) recovering the frozen original cell seeds, and continuously carrying out passage for 10 times to prepare the basic cell seeds. 1 ml/piece, total 120 pieces; working cell bank: the revived basal cells were passaged 10 times in succession, and working cell seeds were prepared at 1 ml/count for a total of 250 counts. The cells of each generation are tested according to the appendix of Chinese veterinary pharmacopoeia, and the cells are free from the pollution of bacteria, mould, mycoplasma and exogenous viruses.

2.4 identification of cell lines

The test is carried out according to the regulation of 'cell standard for production and test' in the attached page of the current Chinese veterinary pharmacopoeia, and the standard is met.

Example 2 preparation of inactivated vaccine against feline panleukopenia Virus

The preparation process of inactivated feline panleukopenia virus vaccine (FPV BJ05C strain) is as follows. From the quality inspection results of 3 batches of vaccines prepared in a laboratory and 5 batches of vaccines prepared in an intermediate trial mode, the quality of vaccine products prepared by the process flow is stable, and the quality inspection results completely meet the established quality standards.

1. Preparation of virus liquid

During virus propagation, a number of factors affect the proliferation of cells and viruses. The FPV replication requires the DNA polymerase of host cells, and the cells with vigorous proliferation capability and mitosis are most suitable for FPV proliferation, so that the cells are inoculated and cultured simultaneously when the FPV is propagated; the serum concentration in the culture solution obviously influences the replication of the virus, and through a series of comparative tests, the cell culture solution with the serum concentration of 10 percent and the maintenance solution with the serum concentration of 2 percent can be used for reproducing the virus solution with stable and high titer; in addition, the results of comparative experiments on the pH value, cell concentration, virus inoculation dose, virus harvesting time and the like of the culture solution show that the pH value of the cell maintenance solution is controlled between 7.2 and 7.4 during virus propagation, and the cell concentration is selected to be 1 multiplied by 10 during virus inoculation⁵～5×10⁵1% (v/v) virus content of 10 per ml cell virus inoculation dose^6.0TCID₅₀The optimal harvest time of the seed virus and the virus with the concentration of more than one ml is 72-96 h, and the virus liquid with high virus content can be obtained.

In this example, a cell culture solution (trade name: 1640, obtained from Gibco) having a serum concentration of 10% and a pH of 7.3, a maintenance solution (trade name: MEM, obtained from Beijing Huabai Tai Biotech. Co., Ltd.) having a serum concentration of 2% and a cell concentration of 3X 10 of F81 at the time of virus inoculation were used⁵One cell per ml, with an inoculum size of 10 virus at 1% (v/v)^6.0TCID₅₀Perml of feline panleukopenia virus FPV BJ05C strain, the virus harvest time was 72 h.

2. Inactivation process

FPV BJ05C is subjected to inactivation comparison tests by selecting formaldehyde solutions with final concentrations of 0.1%, 0.2%, 0.3% and 0.4% and beta-propiolactone with final concentrations of 0.05%, 0.03%, 0.025% and 0.02%, and test results show that the feline panleukopenia virus can be completely inactivated after 0.025% of the beta-propiolactone is treated at 4 ℃ for 48 hours, and the results show that the beta-propiolactone has small damage to antigens, and the toxicity of the beta-propiolactone disappears after hydrolysis at 37 ℃ for 2 hours. The FPV antigen is added into 0.3 percent of formaldehyde solution and inactivated for 48 hours at 37 ℃ to achieve the aim of completely inactivating the virus; however, formaldehyde is highly irritating and causes adverse reactions if free formaldehyde remaining in the vaccine enters animal organisms.

3. Emulsification process

The inactivated feline panleukopenia virus vaccine is prepared by emulsifying commercial aluminum hydroxide adjuvant of American Saimeri Federation company with a final concentration of 1000 mug/ml and inactivated feline panleukopenia virus (FPV BJ05C strain) with a final concentration of 0.025 percent. Before adjuvant use, the aluminum adjuvant bottle was gently shaken. Before emulsification, unfreezing and mixing the inactivated virus liquid and the inactivated virus liquid in the same batch, dripping an aluminum hydroxide adjuvant into the virus liquid under the aseptic condition until the final volume ratio of the adjuvant to the virus liquid is 1:3, and uniformly stirring the mixture at 200r/min by using a homogenizer; in order to stabilize the interface and eliminate bubbles and obtain the best emulsification effect, the mixture needs to be stood for 30min and then subpackaged.

4. Quality standard for semi-finished product inspection

4.1 sterility test

The bacteria-free growth is carried out according to the examination of the appendix of the current Chinese veterinary pharmacopoeia.

4.2 assay of Virus content

The virus seeds were serially diluted 10-fold in DMEM medium, 100. mu.l of each dilution was added to a 96-well cell culture plate in 8 replicates, followed by 100. mu.l of F81 cell suspension per well (cell density 2X 10)⁵/mL DMEM containing 10% newborn calf serum) and normal cell culture as control, at 37 deg.C with 5% CO₂Culturing in an incubator, replacing a maintenance solution of 2% newborn calf serum after 12h, continuously observing for 4-5 days under the same culture condition, observing cytopathic effect (CPE) day by day, and judging CPE if cells shrink, gather and increase particles, and some cells fuse, fall off and have more gaps. The number of cytopathic wells was recorded and TCID was calculated according to the Reed-Muench method₅₀. The results show that the virus solution per milliliter is not less than 10^6.0TCID₅₀。

4.3 inactivation assay

Taking the inactivated virus solution, pressingLight 10⁰、10^-1、10^-2After dilution, the cells were inoculated into 3 flasks of F81 cells (cell density: 3X 10)⁵/ml)，37℃、5％CO₂Culturing and observing, setting virus control, and continuously passaging for 3 generations. The result was no cytopathic effect. All 3 batches of semi-finished products produced in the laboratory are qualified.

5. Quality standard for finished product inspection

5.1 safety inspection Standard

In order to ensure the safety of the vaccine, the safety tests of single dose, repeated single dose and overdose are carried out on 5 batches of prepared vaccines in sequence on healthy kittens, adult cats and pregnant cats. Test results show that single dose and single dose repeat and single overdose inoculation of young cats, adult cats and pregnant cats have no adverse local reactions such as partial infarction, red swelling and the like when injected, and the whole body reactions such as depression, anorexia or abolishment, diarrhea, body temperature rise and the like when pressed; the difference between the total number of leucocytes before and after inoculation of young cats and adult cats is not obvious; the weight gain of the kitten is basically consistent with that of the control group, and all organs are not pathologically changed by the autopsy; pregnant cats are normal in pregnancy and delivery, and have no cases such as abortion, premature birth, dead fetus and mummy fetus. The results indicate that one single dose, one repeat dose, and one overdose of the vaccine are safe for kittens, adult cats, and pregnant cats.

5.2 preparation of efficacy test standards

5.2.1 test of correlation between antibody titer and immune toxicity challenge protection

Respectively immunizing 1 cat leukopenia virus inactivated vaccine (batch No. 201801) qualified in safety test with 5 cats (FPV neutralization titer is not higher than 1:4 or HI antibody is not higher than 1:8) with health susceptibility of 45-60 months age according to different dosages of 0.5 ml/part, 1 ml/part and 2 ml/part, respectively, immunizing with the control group of test animals after 21 days of immunization, respectively collecting blood, measuring the corresponding antibody titer in serum, and simultaneously collecting blood with 10 batches of cat leukopenia virus inactivated vaccines (batch No. 201801)^6.0TCID₅₀The result of continuous observation for 21 days shows that cats with FPV HI less than 1:32 in serum are completely attacked, and cats with FPV HI more than 1:32 are completely resisted by the virulent attack of the FPVBJ05 strain. Immunizing antibodies against vaccinesAccording to the test result of the research on the correlation between the potency and the challenge protection, the HI antibody potency capable of protecting the cat against the FPV virulent challenge is not lower than 1: 32.

5.2.2 minimal immunization dose

3 batches of inactivated vaccines 201801, 201802 and 201803 of cat leukopenia virus, which are qualified in safety tests, are respectively immunized with 10 healthy sensitive kittens (FPV HI antibody is not higher than 1:8) with the age of 45-60 days according to different dosages of 0.5 ml/part, 1 ml/part, 1.5 ml/part and 2 ml/part, 10 non-vaccinations are additionally arranged as controls, blood is respectively collected on the 7 th day, the 14 th day and the 21 th day before and after vaccination, and the hemagglutination inhibition titer of FPV is determined. 21 days after inoculation, the 5 th generation culture of the FPV BJ05 strain is orally taken for virus attack, the continuous observation is carried out for 21 days, and the virus attack protection condition of the test cat is recorded. According to the detection condition of serum antibodies and the protective effect of challenge immunity, the minimum immune dose of the inactivated vaccine is determined to be 1.0 ml/part, and the using dose is determined to be 1.0 ml/part.

5.2.3 correlation of mouse serological potency test with Cat serological potency test

3 batches of inactivated vaccines 201801, 201802 and 201803 of feline panleukopenia virus produced in a laboratory are used for subcutaneously immunizing 5 BALB/c mice 19 to 21 days old and 5 Chinese garden cats 45 to 60 days old according to the dose of 0.05 ml/cat, 0.10 ml/cat, 0.15 ml/cat and 0.20 ml/cat respectively, and the HI antibody titer is determined and the HI antibody correlation of the mice and the cats is analyzed on 7 days, 14 days and 21 days after immunization and a control group. The results showed that, when mice were immunized at 0.1 ml/dose, FPV HI titers were not lower than 1:32, the same immune efficacy as that of the cats immunized with 1.0 ml/dose can be achieved. Therefore, in the process of determining the quality standard of production, the efficacy test of the vaccine can be carried out by adopting a method for detecting the titer of the FPV HI in the serum of the mouse, namely the vaccine is qualified if the titer of the FPV hemagglutination inhibition antibody is not less than 1:32 14 days after the mouse is required to be immunized by the vaccine at 0.1 ml/dose.

6. Test of immune generation period and immune duration

3 batches of inactivated vaccines 201801, 201802 and 201803 with safety tests qualified for feline panleukopenia virus are used for immunizing healthy sensitive cats respectively. In order to further master the immune efficacy and the immune duration, a reasonable immune program is formulated to ensure that the immune cat keeps a high and lasting antibody level, and the antibody detection is carried out on the immune cat at different times so as to master the antibody consumption and growth rule.

The test result shows that the titer of the FPV hemagglutination inhibition in the serum continuously increases along with the time, namely the immunoprotection against the FPV is generated 14 days after immunization, the peak is reached by 2 months, and then the peak is slowly reduced to the protective level. The experimental results are combined, the immune protection period of the vaccine is set to be 6 months, and the non-challenge test result of the protection period shows that the protection period of the vaccine is at least 6 months, so that the vaccine is inoculated once every 6 months to ensure the immune efficacy.

7. Determination of maternal antibody levels and immunization period

The influence of maternal antibodies on vaccine immunity is determined by studying the change of protective antibodies in the serum of immunized kittens at different times after weaning, so as to select proper vaccine immunity time. The result shows that the maternal antibody level of the kitten is still high 1 week after weaning, and the vaccine immune effect is seriously influenced; 2-3 weeks after weaning, the maternal antibody level of the kitten is reduced to a lower level, the influence on vaccine immunity is small, and the immunized kitten can establish better active immunity. Therefore, according to the change rule of the maternal antibody and the change condition of protective antibodies in the serum of the immunized cats at different immunization occasions, the optimal time of vaccine immunization is determined as 14-21 days after weaning.

8. Determination of immunization frequency

The vaccine is used for preventing cat leukopenia, and the immune period is 6 months. 1 ml/mouse, subcutaneous injection. According to the duration of the maternal antibodies of the kitten, the 14-21 days old of the kitten is immunized 1 time after weaning, the boosting immunization is performed 1 time after 2-3 weeks, the kitten is inoculated once every 4 weeks, the immunization is continuously performed 3 times, 1 part of the kitten is inoculated every time, and the kitten is inoculated once every 6 months later.

9. Shelf life of vaccines

The invention carries out experimental study on the storage life of 3 batches of prepared inactivated vaccines (201801, 201802 and 201803), the 3 batches of vaccines are stored for 9, 12, 15 and 18 months at the temperature of 2-8 ℃, and samples are respectively taken at each time period to detect the properties, safety and immune efficacy of the vaccines. The result shows that the characters of 3 batches of vaccines are not obviously changed after being stored for 18 months at the temperature of 2-8 ℃, and the sterility test and the safety meet the requirements of quality standards. In the efficacy test, the HI antibody titer of one mouse is 1:32 after the mice are immunized by 1801 batches of vaccines stored for 18 months. 201801 and 201802 batches of vaccines are stored for 18 months, after the cats are immunized with the cat sample, the HI antibody titer of one cat is 1:32, but the average HI antibody level of the immunized cat is 1:64, and in consideration of titer loss caused by the vaccines in the transportation and use processes, the storage period of the vaccines is set to be 2-8 ℃ for 15 months.

10. Compared with the immunity effect of similar commercial seedlings in China

10.1 comparative test for safety

10 healthy susceptible cats (FPV HI antibody titer is not higher than 1:8) with the age of 45-60 days are randomly divided into two groups A and B, each group comprises 5 animals, the group A is subcutaneously injected with developed FPV BJ05C inactivated vaccine with 1 part (1 ml)/one animal, the group B is subcutaneously injected with 'Miaosan' triple inactivated vaccine (cat rhinotracheitis, mosaic virus disease and aleukocytosis) produced by Shuiteng company, the batch number is 1620390A) with 1 part (1 ml)/one animal, and the 5 healthy susceptible cats are not inoculated to serve as negative controls. Clinical manifestations of vaccinated cats (mental, appetite, body temperature, feces, etc.) were observed locally and systemically for 21 consecutive days to compare the safety of the vaccine. The results show that within 21 days of the observation period, every observation item of the inoculated cat is normal, the two vaccines are safe to the cat, and the two vaccines have no difference in safety.

10.2 comparative test for immune Effect

The FPV BJ05C inactivated vaccine and Miaosan are respectively injected subcutaneously with 5 healthy and susceptible Chinese garden cats (FPV HI antibody is not higher than 1:8) at the age of 45 days, each of the 5 cats is 1 ml/head part, and the 5 healthy and susceptible Chinese garden cats at the age of 45 days are not inoculated as negative control. Blood was collected and serum was isolated at 7 days, 14 days, 21 days, 30 days, 2 months, 4 months, and 6 months before and after immunization, respectively, and the FPV hemagglutination inhibition titer (HI) in the serum of the immunized cat was measured. The results show that the FPV hemagglutination inhibition titer in the serum of the immunized cat reaches a peak about 2 months after the two vaccines are immunized, and the FPV hemagglutination inhibition titer is still maintained at a higher level up to 6 months after the immunization. And within the detection period of 6 months, the FPV HI antibody level of the two antibodies is not significantly different. However, the strain used in the invention is separated after a large amount of epidemiological investigation in a laboratory, and the inactivated vaccine is more matched with the domestic epidemic strain, thereby being beneficial to the prevention and control of the feline panleukopenia.

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

TABLE 1 serum HI titers in immunized cats

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences

<120> feline panleukopenia virus FPV BJ05 strain and application thereof

<130>KHP201110477.3

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>5118

<212>DNA

<213> Feline panleukopenia virus (Feline panleukapia 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 gggaggggaa 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 tcagattttt 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 tttttatgtt 4560

tattacatat caactagcac ctagaaaatt atattaatat acttactatg tttttatgtt 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

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;

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;

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.