CN111635890A

CN111635890A - Dog parvovirus new CPV-2b strain and application thereof

Info

Publication number: CN111635890A
Application number: CN202010494979.8A
Authority: CN
Inventors: 秦彤; 崔尚金; 由欣月
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2020-06-03
Filing date: 2020-06-03
Publication date: 2020-09-08

Abstract

The invention discloses a Canine Parvovirus (Canine Parvovirus, CPV) strain newCPV-2b/YXY/2019, wherein the microorganism preservation number is CGMCC No. 19297. The inactivated vaccine prepared by inactivating the strain has excellent protection level on animals, is obviously superior to commercial vaccine strains, and has huge potential and value for developing veterinary clinical application vaccines.

Description

Dog parvovirus new CPV-2b strain and application thereof

Technical Field

The invention belongs to the fields of microorganisms and bioengineering, and particularly relates to a canine parvovirus (Canineparvovirus, CPV) CPV-2b/YXY/2019 strain and application thereof.

Background

Canine Parvovirus (CPV) is considered to be one of the smallest DNA viruses in the world, and the virus particle is spherical, has a 20-face-symmetric structure, and is about 18-30nm in diameter (RIMMELZWAAN et al, 1991; AGBANDJE et al, 1993), has no envelope coating, and is a single negative strand DNA virus.

CPV was originally isolated in 1976 by Binn in healthy dogs, and the pathogen was called CPV-1, also known as Canine parvovirus (MVC). CPV-2, which is very different from CPV-1, was subsequently isolated in 1977 by two scholars, Eugster and Naiml (Rokorea et al, 1995) from feces of dogs with hemorrhagic enteritis, and reports on CPV-2 were subsequently made in the United kingdom, Germany and other countries (SAGAZIO et al, 1998), and the prevalence of this virus was confirmed in China by Xuhankun in 1983 (Xuhankun et al, 1983). CPV was considered the cause of canine viral diseases in the 70's of the 20 th century, followed the root to its ancestor, which was generally considered to be Feline Panleukopenia Virus (FPV), although there was no evidence to determine whether variation could be achieved by other species of animals for canine infection, CPV-2 could cause hemorrhagic enteritis in canines. Six to seven amino acids between FPV and CPV-2 determine changes in the VP2 domain that are associated with the host cell transferrin receptor (TfR). The higher mutation rate of CPV genes compared to FPV (BATTILANI et al, 2006; DECARO et al, 2009; DECARO et al, 2008; SHACKETON et al, 2005) also constitutes a difference between CPV and FPV, with a mutation rate of about 10 per year at each site^-4Minor substitutions/sites/year (HOELZER et al, 2008 b; PARRISH et al, 1999). After 1978, two new antigenic mutations emerged, which could be distinguished by monoclonal antibodies, CPV-2a, discovered by Parrish et al in 1985, and CPV-2b in 1991 (HOELZER et al, 2008 a; PARRISH et al, 1985), respectively. In CPV-2a, compared with CPV-2, the VP2 gene of CPV-2a is mutated at positions 87(Met → Leu), 101(Ile → Try), 300(Ala → Gly), 305(Asp → Try) and 555 (Val → Ile). Gene sequence analysis shows that most CPV-2a has a 555 bit (Ile → Val) recurrent mutation. Parrish (1991) typed viruses isolated in the last few years with monoclonal antibodies in 1991, showed 1-2 antigenic neutralizing epitope differences between CPV-2a and CPV-2, i.e., a neoantigenic variant CPV-2b was generated. When the difference of the CPV-2a gene sequence is analyzed, it is found that there are mutations of Asn → Asp and Ile → Val at the 426 and 555 amino acid positions of VP2 gene, but CPV-2b can not only infect dog but also at the site of CPV-2aFeline replicative disease is pathogenic to cats (ZHANG et al, 2010). CPV-2a and CPV-2b have been the predominant popular until 2000. In the early 90 s of the 20 th century, mutation at position 297 (Ser → Ala) of VP2 gene resulted in new CPV-2a and CPV-2b strains, namely, new CPV-2a and new CPV-2b variants, and gradually become popular among dogs and cats. Although the 297 (Ser → Ala) variation does not significantly alter the antigenic epitope of the virus, it may affect the affinity of the virus for the host animal recipient due to its spatial structure in close proximity to the capsid protein surface fiber, rendering it immune unprotected (OHSHI et al, 2008; BUONAVOGLIAet al, 2001). A new antigenic CPV was detected in Italy in 2000 (DECARO et al, 2006), and it was found by a series of identification measures that the antigenic CPV differs from CPV-2a and CPV-2b in the two amino acid positions S297A and D426E, and the 426 nd amino acid of the VP2 protein shows Asp-Glu substitution (see

et al, 2007), since this CPV-2c type appeared and prevailed.

The CPV isolate in China also has a genetic variation phenomenon, CPV-2 is popular in the early 80 s of the 20 th century, but CPV separated after 1986 is mainly CPV-2a, and CPV-2a and CPV-2b are widely coexisted. Besides the mutant strains of CPV-2, CPV-2a and CPV-2b, 2new mutant strains which are generated by further evolution based on CPV-2a are found, and the exact biological significance of the mutant strains is not clear. The main current popular are new CPV-2a and new CPV-2 b.

Canine parvovirus is an acute, virulent and highly contact infectious viral disease which is clinically mainly characterized by hemorrhagic enteritis and vomiting and is distributed worldwide, the morbidity is high and can be about 75 percent, and the mortality is about 50 percent. Canine parvovirus, a causative agent of canine parvovirus disease, can infect not only dogs, but also cats and ferrets. The canine parvovirus disease is caused by a plurality of factors, and the disease can be caused by sudden change of the feeding environment. Canine parvovirus can cause hemorrhagic enteritis and suppurative myocarditis symptoms of dogs. The virus is mainly excreted by feces, and the exposure of healthy dogs to the feces of sick dogs increases the infection probability. However, the infected eating and drinking utensils of the sick dog can transmit virus to cause diseases of other dogs. The disease expels toxin more quickly, the animals can discharge the virus 3 days after the natural infection, the animal has stronger infectivity, and typical clinical symptoms can be shown one week after the infection (JOAO et al, 2008).

Due to the rapid variation speed, the immunity effects of the inactivated vaccine and the attenuated vaccine used in the clinical practice are reduced to different degrees, and the existing vaccine can not completely meet the requirements of disease prevention and control and market consumption. Therefore, in order to grasp the variation of the currently circulating strains and thus more effectively prevent and control canine parvovirus disease, there is a continuing need in the art for research on newly isolated CPV strains and use thereof in the development of new canine parvovirus vaccines or therapeutic preparations.

Disclosure of Invention

In order to make up for the defects and shortcomings in the prior art, the CPV virus is obtained by separating CPV confirmed case excrement samples in Beijing area, and identification and animal tests are carried out by various methods. The invention provides a separated canine parvovirus strain and application thereof. The canine parvovirus strain has excellent immunogenicity, and a vaccine prepared by using the canine parvovirus strain has good protective efficacy on canine parvovirus diseases. The purpose of the invention is realized by the following technical scheme:

the invention provides a Canine Parvovirus (CPV) new CPV-2b/YXY/2019 strain, wherein the microorganism preservation number is CGMCC No. 19297; the preservation time is 03/05/2020; the preservation unit: china general microbiological culture Collection center; and (4) storage address: xilu No.1, Beijing, Chaoyang, Beijing, Kyoho, institute for microbiology, China academy of sciences. The VP2 gene sequence of the canine parvovirus strain is shown in SEQ ID NO. 1.

In another aspect, the present invention provides a composition comprising Canine Parvovirus (CPV) new CPV-2b/YXY/2019 strain.

In a specific case, the composition is a vaccine preparation for preventing and/or treating diseases caused by canine parvovirus, and the active component of the vaccine preparation comprises an attenuated and/or inactivated canine parvovirus strain new CPV-2 b/YXY/2019.

In a particular case, the vaccine formulation further comprises a pharmaceutically acceptable carrier mixable with the canine parvovirus strain; and/or, a pharmaceutically acceptable adjuvant; the adjuvant is preferably selected from Freund's adjuvant, aluminum hydroxide, aluminum phosphate, aluminum oxide, saponin, dextran such as DEAE-dextran, vegetable oil (such as peanut oil, olive oil and/or vitamin E acetate), mineral oil, lecithin, bacterial lipopolysaccharide, peptidoglycan, proteoglycan, polyalcohol, propylene glycol, polyethylene glycol, glycerol, ISA720, ISA206, ISA50, ISA51, ISA15AVG, ISA760VG, GEL 01, and propolis.

In another aspect, the present invention provides a method for preparing a canine parvovirus vaccine, which comprises the step of attenuating and/or inactivating a canine parvovirus strain new CPV-2b/YXY/2019 to be a virulent or avirulent strain.

In another aspect, the invention provides a vaccine prepared by the above method.

In another aspect, the present invention provides a method for preparing a canine parvovirus antibody, comprising the steps of immunizing an animal with a canine parvovirus strain new CPV-2b/YXY/2019 as an immunogen to produce a canine parvovirus antibody; the antibody is a neutralizing antibody or a binding antibody; preferably a monoclonal antibody, a polyclonal antibody, a single chain antibody, or a single domain antibody.

In another aspect, the invention provides a virus culture method, wherein a canine parvovirus strain new CPV-2b/YXY/2019 is inoculated into a host cell for culture and/or passage; the host cell is preferably F81.

In another aspect, the invention provides a dog parvovirus strain new CPV-2b/YXY/2019 for use in preparation of a preparation for detecting, preventing and/or treating diseases caused by dog parvovirus; the preparation is preferably a detection kit, a vaccine preparation or an antibody preparation.

In a specific case, the canine parvovirus detection kit comprises an amplification primer for detecting the VP2 gene fragment of the canine parvovirus strain new CPV-2 b/YXY/2019.

In a specific case, the amplification primers of the VP2 gene fragment are shown as SEQ ID NO.2 and SEQ ID NO. 3; or as shown in SEQ ID NO.4 and SEQ ID NO. 5.

In a specific case, the canine parvovirus detection kit comprises a binding antibody of the canine parvovirus strain new CPV-2 b/YXY/2019.

In a particular case, the vaccine formulation is administered by subcutaneous injection, intramuscular injection or nasal drop immunization.

The term "application" or "use" as used herein may refer to both diagnostic or therapeutic applications and non-diagnostic or therapeutic applications, such as scientific research.

The invention has the beneficial effects that: the CPV is separated from excrement of a dog suffering from hemorrhagic enteritis in a certain animal hospital in Beijing area, and a strong virulent strain with strong pathogenicity is obtained by screening. The inactivated vaccine prepared by inactivating the inactivated vaccine has excellent protection level on animals, and is obviously superior to commercial vaccine strains.

Drawings

FIG. 1 CPV infection of F81 cells: (a) negative control, normal F81 cells (200 ×); (b) CPV infected F81 cells (200 ×).

FIG. 2 shows the result of the amplification of the conserved sequence of VP2 gene: m, BM2000+ molecular standard mass; 1. negative control; 2. a fragment of interest.

FIG. 3 shows the amplification results of the full-length sequence of VP2 gene: m, BM2000+ molecular standard mass; 1. a fragment of interest.

FIG. 4 alignment of key amino acid positions.

FIG. 5 phylogenetic analysis tree of canine parvovirus VP2 gene.

FIG. 6. results of indirect immunofluorescence assay (100 ×): (a) CPV seeded F81 cells; (b) f81 cells not inoculated with virus.

FIG. 7 Electron microscopy of CPV isolates: (a) CPV virus particles (42000 ×); (b) CPV virus particles (220000 ×).

FIG. 8 one step growth curve of CPV.

FIG. 9 Experimental clinical symptoms for canine parvovirus animals: (a) the experimental dog is listened; (b) bloody stool; (c) drying the test dog rhinoscope; (d) test dogs had bloody buttock stains.

FIG. 10 results of experimental animal dissection of canine parvovirus: (a) the abdominal cavity; (b) a bowel; (c) each section of intestinal tract.

FIG. 11 dog parvovirus animal test jejunal transection (0.56X): (a) control dog jejunal transection results; (b) test dogs jejunal transection results.

FIG. 12 Experimental cecal sections from canine parvovirus animals: cecum (20 ×); cecum (40 ×).

Detailed Description

The present invention is described in further detail by the following examples, but it should be understood that the present invention is not limited by the following. The methods and procedures not described in detail are performed with reference to experimental methods and procedures conventional in the art.

Example 1: isolation and identification of canine parvovirus CPV-2b strain

1. Separating a sample: animal hospitals confirmed the diagnosis as the fecal swab or anal swab of canine parvovirus positive case, and the CPV simple infection sample is taken for separation after the sample is collected back to the laboratory and is subjected to other pathogenic nucleic acid detection. The main collection area of the disease is concentrated in animal hospitals in Beijing City and Hebei province.

2. Cell:

the canine parvovirus isolation work used F81 cells, which are laboratory-maintained cell lines.

3. Treatment of fecal samples:

and (3) immersing the CPV positive excrement swab into a centrifuge tube filled with 5ml of PBS, slightly smashing the cotton swab repeatedly until the clear liquid is turbid, gradually recovering the white color or slight yellow color of the cotton swab, and bagging and storing the cotton swab after the cotton swab is twisted on the centrifuge tube wall. The suspension in the centrifuge tube was centrifuged at 6000rpm for 6 min. And (4) taking the supernatant, and then discarding the excrement residue, wherein the supernatant is reserved.

4. Passage and culture of cells

Recovery of F81 cells: freezing in liquidTaking out F81 cells in nitrogen, rapidly placing into a 37 ℃ water bath, continuously shaking until the liquid in the freezing tube is completely melted, transferring the cell sap in the freezing tube into a15 ml centrifuge tube in a biological safety cabinet, placing the centrifuge tube into a horizontal centrifuge, balancing, and centrifuging at 1500rpm for 6 min. After centrifugation, visible cells are settled at the bottom of the centrifuge tube, supernatant is carefully sucked and discarded by a pipette gun, and then 5mL of DMEM cell culture medium with fetal bovine serum concentration of 5% is added to blow and evenly mix the settled F81 cells so as to evenly disperse the cells in the culture medium. The cell suspension was then transferred to a T25 cell vial with 5% CO at 37 deg.C₂After culturing for 12h under the culture condition, discarding the original culture medium in the culture bottle, washing the culture bottle once with PBS, changing the culture bottle into a DMEM cell culture medium containing 2% fetal calf serum, continuously culturing until the cell monolayer is fully paved in the cell bottle, and then carrying out cell passage.

Passage of F81 cells: in a biosafety cabinet, medium in the flask was removed by pipetting, the full monolayer of F81 cells was washed with 1mL PBS, the PBS soaked cell monolayer was shaken 3-5 times and the PBS was removed and repeated once. Adding 500 mu L of 0.25% EDTA trypsin containing phenol red for digestion, shaking the trypsin for 3-7s, then removing the trypsin, horizontally standing the cell bottle mouth for 30-60s by passing a fire cover, and immediately adding 10mL of DMEM cell culture medium with fetal calf serum concentration of 5% to stop digestion and repeatedly blowing and uniformly mixing the cell culture medium with a pipette gun when the cell bottle is shaken slightly to see that the cells at the bottom of the bottle flow in a quicksand shape, so that the cells are dispersed into uniform cell suspension. Pipette 5mL of the cell suspension into a new vial of T25 cells, leave the remaining 5mL in the original vial, and place the two vials of cell caps at 37 ℃ in 5% CO₂Culturing in a constant temperature incubator to obtain a new generation of F81 cells.

5. Isolation and passage of viruses

The grown F81 monolayers were digested as described above in step 4 and then passaged in DMEM cell culture medium containing 2% fetal bovine serum. The treated sample supernatant was filtered through a 0.22 μ M filter and simultaneously seeded at 4% of culture volume into the freshly passaged nonadherent suspension of F81 cells. Lightly blowing, mixing, covering with a cover, and placing at 37 deg.C with 5% CO₂The constant temperature incubator is used for static culture. Every 12h watchThe cell state was observed once, and the observation was stopped until 72 hours. And (4) when the canine parvovirus typical cytopathic effect does not exist after 72h of culture, freezing and thawing the cells for harvesting, and then performing virus inoculation according to the same virus inoculation mode, wherein the process is blind transfer. After the blind passage 5, the separation was considered negative if no cytopathic effect appeared. If cytopathic effect occurs, the virus is harvested when the cytopathic effect reaches 80%, and the virus can be passaged according to the synchronous virus inoculation step after repeated freeze thawing for three times.

The supernatant was aseptically filtered through a 0.22 μ M filter and then inoculated to F81 cells simultaneously, while a non-inoculated negative control was set. After two blind generations, the isolate begins to have cytopathic effects (CPE) such as net pulling and shedding when being cultured for 48h, and the virus is continuously transmitted to the eighth generation as shown in figure 1, so that the isolate is determined to have stable cytopathic effects.

6. Viral identification

(1) And (3) PCR identification:

the primers are identified to amplify 1755bp and 574bp respectively aiming at the canine parvovirus VP2 gene. The primers were synthesized by Beijing Liuhe Huada Gene science and technology Co. The primer sequences are shown in table 1 below:

TABLE 1 CPV PCR identification primer List

Because the canine parvovirus nucleic acid is DNA, the viral genome DNA rapid extraction kit of the Edley Biotechnology Ltd is selected to extract the canine parvovirus DNA. The operation steps are carried out according to the instruction of the virus genome DNA rapid extraction kit.

The extracted virus DNA is used as a template to carry out common PCR amplification, and the reaction system and the reaction program of the amplification are 1)574bp fragments, namely a reaction system of 2 × Taq PCR MasterMix 10 mu L, P3P 4 each 1 mu L, a DNA template 2 mu L, ddH₂Make up to 20. mu.L of O. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 4min, 30 cycles: denaturation at 95 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 35s, re-extension at 72 deg.C for 10min, and storing at 4 deg.C.

2)1755bp fragment, reaction system including LA Taq enzyme 0.5 μ L, 10 × LA Buffer II 5 μ L, dNTP8 μ L mix, 2 μ L DNA template, 1 μ L each of P1 and P2, ddH₂O is complemented to 50 mu L, the reaction condition is that 94 ℃ is pre-denatured for 1min, 35 cycles are that 94 ℃ is denatured for 30s, (55 ℃ to 70 ℃) gradient annealing for 30s, 72 ℃ is extended for 2min, 72 ℃ is extended for 10min, 4 ℃ is stored, the reaction condition is determined after the optimal annealing temperature is determined, the product amplified by 2 × Taq PCR MasterMix can be directly subjected to gel electrophoresis, the product amplified by LA Taq enzyme needs 10 mu L6 × DNA/RNA Loading Buffer to be uniformly mixed and subjected to electrophoresis observation, the product obtained by PCR is subjected to electrophoresis for 33min under the condition that the voltage is 120V, agarose gel is placed under a gel imaging system to observe whether the product has bands and the band size, and the long band is photographed for gel cutting recovery and sequencing.

The result is shown in FIG. 2, which is a 574bp fragment amplified against the CPV VP2 gene conserved sequence, and the amplification result is consistent with the expected size. And then amplifying the VP2 gene full length to obtain a 3 picture, wherein the fragment length is 1755bp, the size is consistent with the expected size, bands with the expected size are obtained by two times of amplification, the isolate is canine parvovirus, and 1755bp products are recovered by glue and then sent to Beijing Liuhe Huada GenBank for sequencing.

After aligning the sequencing return result with VP2 genes of other CPVs logged on GenBank, the 426 th amino acid site of VP2 protein of the isolated strain is aspartic acid (D), 297 th amino acid site is alanine (A), which is consistent with the new CPV-2b reference strain, and 426 nd amino acids of CPV-2a and CPV-2c are asparagine (N) and glutamic acid (E), respectively. By aligning the 297 th amino acid and the 426 th amino acid, the canine parvovirus isolate can be determined to be a new CPV-2b type strain, and the figure 4 shows.

The isolated strain and the VP2 gene of the CPV reference strain registered in GenBank are subjected to construction of an evolutionary tree by using biological software MEGA 6.0. As shown in FIG. 5, it can be seen from the results of the VP2 gene clade that the present isolate is different from the newCPV-2b type (M74849), the CPV-2a type (AFK 66757.1) and the CPV-2c type (MK 332007) and the like) on the same branch as the newCPV-2b type strains (AXR98582, KR611461 and the like).

(2) Indirect immunofluorescence assay:

f81 cells growing to a monolayer in a T25 cell bottle are digested according to a method of F81 passage, then the cells are evenly blown and stirred by 10mL of DMEM cell culture medium with fetal bovine serum concentration of 2% to enable the cells to be evenly dispersed in the culture solution, and then F81 cell suspension is transferred into a 96-well plate, wherein each well is 100 mu L. The canine parvovirus isolate virus fluid was simultaneously inoculated into F81 cells. The 96-well plate is placed at 37 ℃ and 5% CO₂After the culture is carried out for 36 hours in a constant temperature incubator, an indirect immunofluorescence test is carried out. Discarding the culture solution in 96-well plate, spin-drying slightly, then slowly adding PBST, slightly shaking for 3-5min, discarding, repeating for 2 times, adding 4% paraformaldehyde which is cooled, and fixing the cells for 30 min. The paraformaldehyde fixing solution is discarded, washed 3 times with PBST, 3-5min each time, and gently patted dry. Blocking with 5% skimmed milk at 37 deg.C for 1 h. The blocking solution was discarded and washed 3 times with PBST, once for 3-5 min. According to the following steps of 1: 1000 as primary antibody, diluting the mouse anti-canine parvovirus monoclonal antibody with PBST, adding a proper amount of the primary antibody into a pore plate, and incubating for 1h at 37 ℃. Discard primary antibody, wash with PBST 3 times, each for 3-5 min. The light-avoiding condition is as follows: 1000 proportion of PBST to goat anti-mouse antibody as a secondary antibody, and adding a proper amount of the secondary antibody into a pore plate to incubate for 1h at 37 ℃. And (4) discarding the secondary antibody under the condition of keeping out of the light, and washing with a proper amount of PBST for 3 times, wherein the washing is carried out for 3-5min once. Fluorescence was observed under an inverted fluorescence microscope. Meanwhile, non-toxic cells were used as negative control.

As shown in FIG. 6, in the inverted fluorescence microscope, the cells that were inoculated with the virus for 36h emitted specific green fluorescence by the excitation light, while the cells that were not inoculated did not fluoresce under the excitation light, which indicates that the experiment is in progress. The isolate can react with CPV antibody specifically, and specific green fluorescence can be seen under an inverted fluorescence microscope, thus proving that the isolate is CPV indeed.

(3)TCID₅₀The determination of (1):

in a biosafety cabinet, F81 cells grown to a monolayer in a T25 cell culture bottle are digested according to a passage method and blown and uniformly mixed by 10mL of DMEM cell culture medium with fetal bovine serum concentration of 2% to uniformly disperse the cells in a culture solution for later use. And continuously diluting the virus liquid by 10 times in a sterilization tube, namely adding 900 mu L of serum-free DMEM culture medium into ten sterilized 1.5mL centrifuge tubes marked with No. 1-10, sucking 100 mu L of the serum-free DMEM culture medium by using a pipette gun, repeatedly pumping and uniformly mixing the 100 mu L of serum-free DMEM culture medium and 900 mu L of serum-free DMEM culture medium in the first centrifuge tube, sucking 100 mu L of serum-free DMEM culture medium from the first centrifuge tube into a 1.5mL centrifuge tube filled with 900 mu L of serum-free culture medium in the second centrifuge tube, and repeating the steps until reaching the tenth centrifuge tube to prepare the virus liquid diluted by 10 times.

The 96-well plate had 12 columns, and 8 wells of each column were used as 8 replicates of one virus dilution. Adding 100 μ L of cell suspension and corresponding dilution of virus diluent 100 μ L into 1-10 rows of 96-well plate, adding 100 μ L of cell suspension and serum-free DMEM 100 μ L into 11-12 rows of 96-well plate, and standing at 37 deg.C with 5% CO₂Culturing for 5-7 days in a constant temperature incubator, observing and recording cytopathic conditions day by day, and finally calculating the TCID of the virus according to a Reed-Muench method₅₀。

In a biological safety cabinet, absorbing 100 mu L of the 3 rd generation virus liquid, diluting the virus liquid ten times, and then performing synchronous inoculation on the virus liquid to perform TCID₅₀The measurement was carried out by observing the number of wells with more than 50% of cytopathic effect after 5 days, and calculating the content of the third generation virus liquid of the isolate to 10 according to the Reed-Muench method^7.3TCID₅₀/mL。

(4) Morphological identification:

after the F81 cells are subcultured according to the subculture step, CPV virus isolate is synchronously inoculated according to 4% of culture volume, and 5% CO is carried out at 37 DEG C₂Culturing in a constant temperature incubator. When F81 cells have 80% lesions, the virus is harvested by repeated freezing and thawing three times, stored in a centrifugal tube for standby, and concentrated when the virus amount reaches 100 mL. Centrifuging the frozen and thawed virus solution in a centrifuge tube at 6000rpm for 40min, taking the supernatant, transferring to a new centrifuge tube, and concentrating the virus by using an ultra-high speed centrifuge.

The concentration method comprises the following steps: preparing a sucrose solution with the concentration of 20% by using PBS, filtering the prepared sucrose solution into a new container by using a 0.22 mu M filter, wherein the sucrose solution needs to be prepared as it is. Adding about 30mL of virus liquid into an ultra-high-speed centrifuge tube, connecting the ultra-high-speed centrifuge tube with a syringe and a long needle, slowly injecting a sucrose solution with the concentration of 20% into the ultra-high-speed centrifuge tube by using the long needle, and covering the bottom of the ultra-high-speed centrifuge tube with the sucrose solution with the dosage of 6-10mL, wherein colorless sucrose and colored virus liquid can be clearly layered. After being strictly balanced by an analytical balance (error is +/-0.005), the mixture is placed into an ultra-high speed centrifuge and centrifuged for 1.5h at 170000 g. And after the centrifugation is finished, the centrifugal tube is stably taken out, then the supernatant is discarded, the centrifugal tube is inverted on the tube frame, and the liquid does not need to flow backwards. The inner wall of the centrifuge tube was wiped dry with a paper towel, each tube was resuspended in 50. mu.L of Ph7.4 PBS and pelleted, and blown repeatedly to disperse the virus particles uniformly. Avoid generating bubbles as far as possible, and reserve.

Preparing a negative staining electron microscope sample: 1. glow discharge: placing the Grid, GIG (Cu 400mesh) on the electron microscope in a glass culture dish, laying the carbon film on the front side of the glass culture dish, and putting the whole glass culture dish into a plasma cleaning machine, wherein the carbon film is upward. Vacuumizing for 3 minutes, and discharging for 30s in a Low gear. This step is an air discharge that negatively charges Grid. 2. Negative dyeing: one end of the Grid was grasped with self-locking forceps and placed flat, and 4. mu.l of the protein sample was incubated on the surface. After 1min, excess liquid was washed off with filter paper and incubated in 2% uranyl acetate. Uranyl acetate was incubated in each droplet of uranyl for 10 seconds using a three-drop method with 33 μ l of each drop and grid. 3. And (4) absorbing and removing redundant uranyl acetate by airing, and naturally airing to observe the normal-temperature rod electron microscope sample.

Synchronously inoculating 3 rd generation virus liquid of a CPV isolate to F81 cells, performing amplification culture to obtain 100mL of 4 th generation virus liquid of the isolate, concentrating by using an ultrahigh-speed centrifugal concentration method, performing phosphotungstic acid negative staining, and respectively amplifying by 11 ten thousand times and 22 ten thousand times under a transmission electron microscope for observation, wherein the result is shown in figure 7, the diameter of virus particles of the isolate is about 26nm, the virus particles are of a three-dimensional symmetric icosahedron, are spherical, are not wrapped by a capsule membrane around the virus particles, and have morphological characteristics basically consistent with the reported shapes of dogs and other parvoviruses.

(5) Measurement of one-step growth curve:

after F81 cells are subjected to subculture digestion, parvovirus is synchronously inoculated into cell suspension, and then the parvovirus is subpackaged into a new T25 cell bottle and placed in 5% CO at 37 DEG C₂The constant temperature incubator is used for culture, and then the culture medium is respectively harvested at the time of 2h, 4h, 8h, 12h, 24h, 36h, 48h, 60h and 72hObtaining a bottle of virus, repeatedly freezing and thawing for three times, and then carrying out TCID on the bottle of virus₅₀And a growth curve was plotted using Excel. The remaining virus liquid is stored at-80.

Measuring TCID by taking virus liquid at 2h, 4h, 8h, 12h, 24h, 36h, 48h, 60h and 72h after inoculation₅₀LgTCID of₅₀The results of the concentration of the virus particles in the sample at a concentration of 3.13, 3.4, 3.75, 4.83, 6.09, 6.29, 6.49, 6.76 and 6.19 μ L were plotted on the ordinate and the inoculation time was plotted on the abscissa, thereby measuring the growth curve in one step. As can be seen from the curve results (FIG. 8), the proliferation rate of the virus is relatively slow within 8h before virus inoculation, the proliferation of the virus is in logarithmic phase within 12h-24h after virus inoculation, and cells begin to appear pathological changes within 24h-48h, namely, the number of cells is small or large. After 60h, the virus content tends to be stable and even tends to decline, and the cytopathic effect reaches 80% -90% so that the virus can be harvested.

(6) Partial study of biological Properties

1) Study of Heat resistance

Taking 3mL of third-generation virus liquid out of a biological safety cabinet, placing the third-generation virus liquid into a15 mL centrifuge tube, heating the third-generation virus liquid in water bath at 50 ℃ for 1h, and treating the third-generation virus liquid with TCID₅₀And (4) carrying out measurement.

2) Study of acid resistance

Absorbing 6mL of third-generation virus liquid in a biological safety cabinet, placing the third-generation virus liquid in a50 mL centrifuge tube, adjusting the pH value of the virus liquid to 3 by using 0.1mol/L hydrochloric acid under the indication of an acidimeter, adjusting the pH value to 7.2 after acting for 2h at room temperature, and adjusting the TCID of the virus liquid₅₀And (4) carrying out measurement.

3) Study of alkali resistance

Sucking out 6mL of third-generation virus solution from biosafety cabinet into a50 mL centrifuge tube, adjusting pH of the virus solution to 8 with 0.1mol/L sodium hydroxide under the instruction of an acidimeter, adjusting pH value of the virus solution to 7.2 after 2 hours of action at room temperature, and adjusting TCID of the virus solution₅₀And (4) carrying out measurement.

4) Ultraviolet irradiation test

And (3) sucking 1mL of third-generation virus liquid in a biological safety cabinet, placing the third-generation virus liquid in a sterilized 1.5mL centrifugal tube, and turning on an ultraviolet sterilizing lamp in the biological safety cabinet for 1 h. Then to its TCID₅₀And (4) carrying out measurement.

As a result: treating TCID with ultraviolet radiation, acid and alkali, and heat treatment₅₀The results of the measurements were 10 respectively^2.62/0.1mL、 10^6.750.1mL and 10^6.64/0.1mL、10^6.20.1mL, acid base and thermally treated TCID₅₀The magnitude of change of the strain is not more than 2, which indicates that the physical and chemical properties of the isolate are relatively stable and the isolate has stronger resistance to the external environment, but the TCID is treated by ultraviolet rays₅₀The obvious reduction indicates that the isolate is sensitive to ultraviolet rays.

The separated Canine Parvovirus (Canine Parvovirus, CPV) is named as new CPV-2b/YXY/2019 strain, and the microorganism preservation number is CGMCC No. 19297; the preservation time is 03/05/2020; the preservation unit: china general microbiological culture Collection center; and (4) storage address: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

Example 2: animal infection test

1. The test animal is 2-3 month old puppy, no breed, and native dog. The puppy is fed for ten days before the test, pathogen detection is carried out simultaneously, the fact that the puppy does not have viral and bacterial diseases is determined, the body surface and the mental state of the puppy are observed, and the fact that the puppy can be tested is determined. After ten days of feeding, the purpose is to reduce the influence of the stress phenomenon of the test dog on the test result, no disease attack occurs within ten days, and the infection test of the canine tiny isolate animal is carried out after the canine tiny detection is negative.

The test groups of test dogs No.1, 2, 3, and 4 were divided into two groups according to body weight, i.e., intramuscular injection group (dogs No.1 and 2) and oral administration group (dogs No.3 and 4). The intramuscular injection group No.1 dog had bloody stool phenomenon in 17h, and both dogs had tomato juice-like bloody stool in less than 24h and were accompanied with strong fishy smell, and the symptoms of abstinence of appetite water, recumbency, listlessness, dryness of nasoscope, bloody filth around hip and anus and no response to call were accompanied with death in 28h and 34h, respectively. The oral group of dogs 3 showed loose stool and bloody stool in about 69h, dogs 4 showed tomato juice-like bloody stool in 80h, and dogs 70h or so showed death in 119h and 125h, respectively, due to abstinence of appetite water, sleepiness, listlessness, call non-response, and the like, as shown in FIG. 9.

2. C, performing dissection to examine pathological changes:

body surface appearance: the subcutaneous dehydration of the body surface of the dead dog is different, the oral test dog has more serious dehydration than the intramuscular injection test dog, and blood stain is adhered around the anus. The eye and the conjunctiva are pale and the gum is pale.

C, performing autopsy: the abdominal cavity is cut open, and a small amount of liquid is accumulated in the abdominal cavity. The eye can show extensive and diffuse bleeding in the intestinal tract, and the pathological changes are mainly seen in the intestinal tract, which shows that the intestinal tract is full of blood stains. The intestinal wall of the intestine becomes thinner and bleeding is more severely concentrated in the middle and rear small intestine, i.e., the jejunum and ileum. The serosa is dark red, and congestion and bleeding are observed under the serosa. Breaking the intestinal cavity, the filth flows out and can smell a strong fishy smell in the form of tomato juice. When the intestinal canal is taken out, the intestinal wall of each segment of the intestinal canal is thinned, bleeding and the like. Mucosa exfoliation and villous atrophy. Mesenteric lymph nodes were engorged, bleeding, swollen, see fig. 10. No pathological changes are visible to the naked eye in other organs.

3. Pathological change of tissue sections:

by the steps of intestinal tissue sampling, fixing by 4% paraformaldehyde, wax block embedding, slicing, HE staining and the like, pathological changes of intestinal tissues of the sick dogs are observed. From FIG. 11, it can be seen that the jejunum with the most severe bleeding has thinner intestinal wall, the intestinal villi is also atrophied, and the presence of distinct red blood cells in the intestinal villi part indicates the bleeding of the jejunum in the test dog. As can be seen by the tissue section of fig. 12: the caecum mucosa crypt epithelial cells are severely necrotic and sloughed off, remaining in the glandular cavity, causing the enlargement of the acinus, which contains necrotic cellular debris. The mucous membrane lamina propria has a small amount of lymphocyte infiltration, the mucous membrane lamina propria and the submucosa show connective tissue hyperplasia, and intestinal villi are seriously shed, which are very anastomotic with hemorrhagic enteritis caused by virus canine parvovirus.

Example 3: virus testing

1. And (3) exogenous virus detection: through inspection, after the 15 th generation toxin of the isolated new CPV-2b/YXY/2019 strain is neutralized by CPV positive serum, no cytopathic effect is generated, and no erythrocyte adsorption phenomenon exists, while the F81 cells and canine kidney primary cells of a control group have CPE and erythrocyte adsorption phenomenon. The new CPV-2b/YXY/2019 virus strain is shown to be free of other foreign viruses.

2. And (3) bacteria inspection: two T.G vials and G.A slants were inoculated with 0.2 mL/vial of cell culture virus. Culturing at 37 deg.C; one of the two virus liquid is cultured at 25 ℃ and observed for 3-5 days, no bacteria grow, and the separated virus liquid is free from foreign bacteria pollution.

3. And (3) mycoplasma test: inoculating 5mL of virus solution into mycoplasma liquid culture medium, culturing at 37 deg.C, transplanting 0.2mL of virus solution onto liquid culture medium and agar plate on

days

1, 5, 15, and 20, respectively, and placing at 37 deg.C and 5% CO₂Incubation was performed at ambient 37 ℃. And checking the agar plate under a low power microscope every 5-7 days, and stopping observation after 14 days of continuous observation when no bacterial colony exists. The liquid culture was observed daily for the presence or absence of reddening or yellowing. If there was no change, observation was stopped at 14 days after the last transplantation of the tubules; if the pH value changes to +/-0.5, the agar plate culture colonies are immediately inoculated. And simultaneously setting an uninoculated negative control group and an inoculated mycoplasma hyorhinis positive control group. New CPV-2b/YXY/2019 group did not have typical "omelet" mycoplasma colony formation, indicating that the isolated virus fluid was free of mycoplasma contamination.

4. Virus proliferation stability test TCID was measured for the 10 th, 20 th and 30 th passages of the new CPV-2b/YXY/2019 strain on F81 cells₅₀And simultaneously carrying out a genetic stability test. The experimental results (see Table 2) show that the new CPV-2b/YXY/2019 strain still maintains high proliferation power when the strain passes through 30 generations.

Table 2 results of experiments on proliferation potency and genetic stability of new CPV-2b/YXY/2019 strain

Example 4: preparation of inactivated vaccine

The method comprises the following steps:

(1) culturing F81 according to conventional method, taking content of 10⁵TCID₅₀Inoculating the New CPV-2b/YXY/2019 strain of the canine parvovirus per mL to F81 cells according to the volume percentage of 5%, adsorbing for 1 hour at 37 ℃, discarding the adsorption solution, and supplementing DMEM containing 10 mu g/mL trypsin as a maintenance solution to continue culturing;

(2) after virus inoculation, recording the condition of cytopathic effect (CPE), harvesting when the cytopathic effect rate reaches more than 80%, repeatedly freezing and thawing at-80 ℃ for three times, centrifuging at 8000rpm for 15min, and collecting supernatant, namely virus liquid; concentrating the prepared virus liquid by 20 times, adding beta-propiolactone according to the proportion of 1:2000, thoroughly mixing, inactivating for 24 hours at 4 ℃, and bathing for 2 hours at 37 ℃ to obtain the stock solution of the canine parvovirus new CPV-2b/YXY/2019 strain vaccine.

(3) And uniformly stirring and mixing the canine parvovirus vaccine stock solution and the sterilized Montanide ISA15AVG adjuvant in a ratio of 1:9 in a sterile environment to obtain the inactivated vaccine.

(4) The exogenous viral, bacterial and mycoplasma assays were negative as described in example 3.

Example 5: inactivated vaccine efficacy test

1. The method comprises the following steps:

(1) the experimental animal is a beagle dog with the age of 30-50 days, and CPV pathogen is detected; and collecting blood in the groin, placing for 1h at 37 ℃, centrifuging for 15min at 1000rpm after coagulation to prepare serum, adopting a neutralization test to measure CPV neutralizing antibodies in the serum, and selecting 15 dogs with negative CPV pathogens and neutralizing antibodies to carry out an immune test of the vaccine.

(2) Immunization groups are as in table 3:

TABLE 3 immunization groups

(3) The immunization method comprises the following steps:

the animals in each vaccine test group were inoculated with 1ml of vaccine subcutaneously in the neck and back, and each immunization was boosted once on days 21 and 42 after the first vaccination. Blood is collected from each beagle dog 7 days, 21 days, 28 days, 42 days, 49 days, 120 days and 180 days after the first inoculation, and serum is separated for antibody titer and titer determination.

2. As a result:

(1) detection results of CPV neutralizing antibodies in serum after vaccine immunization: the standard positive serum, the negative serum and the serum sample to be tested were inactivated at 56 ℃ for 30min for neutralization test. And (3) continuously diluting the inactivated serum by 2 times by using a cell culture medium, sucking 100 mu L of serum of each dilution, adding the serum into a 24-well plate, setting 3 repeated wells for each dilution, adding CPV, slightly shaking and mixing, and then incubating for 1h at 37 ℃. The digested F81 cell suspension was then added at 500. mu.L per well, while serum, virus and normal cell controls were set. After incubation at 37 ℃ for 5 days in an incubator, CPE was observed under a microscope and more than half of the cells were positive in the wells with lesions. The results are shown in Table 4, and the mean values of neutralizing antibody titers of the new CPV-2b/YXY/2019 test groups at 7, 21, 28, 42, 49, 120, and 180 days are respectively: 1:2^8.8、1:2^7.2、1:2^10.8、1:2^9.4、1:2^11.4、1:2^8.4And 1:2^7.8；

The mean values of the neutralizing antibody titers of the fine test groups were: 1:2^8.6、1:2^7.2、1:2^10.6、1:2⁹、 1:2^11.2、1:2^8.25And 1:2^7.5。

TABLE 4 neutralizing antibody titers comparison (mean)

(2) Survival rate after challenge, namely performing challenge experiment by using CPV strength 14 days after three times of immunization (namely 56 days after first inoculation), wherein 1mL of CPV standard strain culture solution is orally taken, and the virus content is more than or equal to 1 × 10⁵TCID₅₀and/mL. The onset and survival of each group after challenge were observed and the results are shown in Table 5 (time is days after challenge). Therefore, the new CPV-2b/YXY/2019 inactivated vaccine has the protection effect obviously superior to that of a commercial vaccine group, realizes full protection on experimental animals, and has the survival rate of 100 percent.

TABLE 5 survival rate after challenge

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, but rather as the subject matter of the invention is to be construed in all aspects and equivalents thereof.

SEQUENCE LISTING

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

<120> dog parvovirus new CPV-2b strain and application thereof

<130>CP120020353C

<160>5

<170>PatentIn version 3.3

<210>1

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<213> complete nucleotide sequence of VP2

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atgagtgatg gagcagttca accagacggt ggtcagcctg ctgtcagaaa tgaaagagct 60

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aatactatgc catttactcc agcagctatg agatctgaga cattgggttt ttatccatgg 600

aaaccaacca taccaactcc atggagatat tattttcaat gggatagaac attaatacca 660

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tttgctacag gaacatttta ttttgattgt aaaccatgta gactaacaca cacatggcaa 840

acaaatagag cattgggctt accaccattt ctaaattctt tgcctcaagc tgaaggaggt 900

actaactttg gttatatagg agttcaacaa gataaaagac gtggtgtaac tcaaatggga 960

aatacaaaca ttattactga agctactatt atgagaccag ctgaggttgg ttatagtgca 1020

ccatattatt cttttgaggc gtctacacaa gggccattta aaacacctat tgcagcagga 1080

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atagcacatc aagatacagg aagatatcca gaaggagatt ggattcaaaa tattaacttt 1260

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ggaattaact atactaatat atttaatact tatggtcctt taactgcatt aaataatgta 1380

ccaccagttt atccaaatgg tcaaatttgg gataaagaat ttgatactga cttaaaacca 1440

agacttcatg taaatgcacc atttgtttgt caaaataatt gtcctggtca attatttgta 1500

aaagttgcgc ctaatttaac aaatgaatat gatcctgatg catctgctaa tatgtcaaga 1560

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ggaattctta gtataatttt ctaggtgcta gtt 33

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Claims

1. A Canine Parvovirus (Canine Parvovirus, CPV) strain new CPV-2b/YXY/2019 has a microorganism preservation number of CGMCC No. 19297.

2. The canine parvovirus strain of claim 1, wherein the gene sequence of VP2 is set forth in SEQ ID No. 1.

3. A composition comprising the canine parvovirus strain of claim 1 or 2.

4. A vaccine formulation for preventing and/or treating diseases caused by canine parvovirus, characterized in that its active ingredient comprises the attenuated, and/or inactivated canine parvovirus strain CPV-2b/YXY/2019 of claim 1 or 2.

5. The vaccine formulation of claim 4, further comprising a pharmaceutically acceptable carrier mixable with the canine parvovirus strain; and/or, a pharmaceutically acceptable adjuvant; the adjuvant can be selected from Freund's adjuvant, aluminum hydroxide, aluminum phosphate, aluminum oxide, saponin, dextran such as DEAE-dextran, vegetable oil (such as peanut oil, olive oil and/or vitamin E acetate), mineral oil, lecithin, bacterial lipopolysaccharide, peptidoglycan, proteoglycan, polyalcohol, propylene glycol, polyethylene glycol, glycerol, ISA720, ISA206, ISA50, ISA51, ISA15AVG, ISA760VG, GEL 01, and propolis.

6. A method for preparing a canine parvovirus vaccine, comprising attenuating and/or inactivating the canine parvovirus strain of claim 1 or 2 to a low-virulent or non-virulent strain.

7. A vaccine prepared according to the method of claim 6.

8. A preparation method of a canine parvovirus antibody is characterized by comprising the following steps: immunizing an animal with the canine parvovirus strain of claim 1 or 2 as an immunogen, wherein the antibody is a neutralizing antibody or a binding antibody; preferably a monoclonal antibody, a polyclonal antibody, a single chain antibody, or a single domain antibody.

9. A method for culturing a virus, comprising inoculating the strain of claim 1 or 2 into a host cell, and culturing and/or passaging the cell; the host cell is preferably F81.

10. Use of the canine parvovirus strain of claim 1 or 2 for the preparation of a formulation for the detection, prevention and/or treatment of diseases caused by canine parvovirus; the preparation is preferably a detection kit, a vaccine preparation or an antibody preparation; preferably, the vaccine formulation is administered by subcutaneous injection, intramuscular injection or nasal drop immunization.