CN108018261B - Canine parainfluenza virus strain and application thereof - Google Patents

Canine parainfluenza virus strain and application thereof Download PDF

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CN108018261B
CN108018261B CN201610946235.9A CN201610946235A CN108018261B CN 108018261 B CN108018261 B CN 108018261B CN 201610946235 A CN201610946235 A CN 201610946235A CN 108018261 B CN108018261 B CN 108018261B
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canine
parainfluenza virus
canine parainfluenza
antigen
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CN108018261A (en
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田克恭
刘玉秀
刘彩红
孙进忠
张许科
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LUOYANG HUIZHONG BIOTECH Co.,Ltd.
Pulaike Biological Engineering Co Ltd
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Luoyang Huizhong Biotech Co ltd
Pulaike Biological Engineering Co Ltd
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
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    • C12N2760/18011—Paramyxoviridae
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    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
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    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
    • C12N2760/18634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The canine parainfluenza virus HeN0718 strain has strong toxicity and good immunogenicity, and after being used for immunizing animals, a vaccine composition prepared from the inactivated antigen can enable animal bodies to quickly generate antibodies, so that the current canine parainfluenza related diseases can be effectively prevented and/or treated. The vaccine composition has good safety and can be used in combination with other antigens.

Description

Canine parainfluenza virus strain and application thereof
Technical Field
The invention belongs to the technical field of biological products for veterinary use, and particularly relates to a canine parainfluenza virus strain, a vaccine composition thereof, a preparation method and application thereof.
Background
Canine Parainfluenza virus (CPIV) is a member of Paramyxoviridae, Paramyxovirinae, and the genus mumps, is an nonsegmented single-stranded minus-strand RNA virus, and CPIV particles under an electron microscope are variable in morphology, generally circular in shape and 80-200 nm in diameter, but are often irregular in morphology due to capsular membrane breakage and present as virus particles with polymorphism.
CPIV has a broad host spectrum, including dogs, guinea pigs, hamsters, mice, tigers, racoon dogs, minks, etc., and often causes bronchitis and bronchopneumonia in dogs, and damages airway epithelial cells. After the dogs are infected with CPIV, the virus can be detected from respiratory secretions, virus particles are infected to healthy dogs through effective gas particles, foams and air, outbreaks are often caused by dog groups with high density, various types of dogs can be infected, the susceptibility of young dogs is higher, and the development of the canine industry of various countries in the world is seriously influenced.
The vaccine is the main measure for preventing and controlling the disease, as early as 1970, Emeay et al developed CPIV D-008 attenuated vaccine, and intramuscular or subcutaneous vaccination can produce neutralizing antibodies, and the use of the vaccine can reduce the incidence of the disease and alleviate clinical symptoms, but can not completely resist infection. There are several companies that are currently providing vaccines containing CPIV at home and abroad, but in clinical practice it has been found that some of the CPIV infection positive dogs have been rigorously vaccinated with a CPIV vaccine history. On the other hand, the attenuated vaccine has the risks of virulence reversion and toxin dispersion in the using process, the virulence attenuation of the existing separated virulent strain is obvious in the culture process, and the prepared inactivated vaccine has low immune potency, so that no related inactivated vaccine product exists at present. Therefore, the CPIV wild strain which is epidemic in China and has good immunogenicity needs to be separated out, and the CPIV wild strain is prepared into a high-efficiency inactivated vaccine to prevent and/or treat the spread of the canine parainfluenza epidemic situation, so that the CPIV wild strain has a far-reaching practical significance.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a canine parainfluenza virus strain, an inactivated vaccine prepared from the canine parainfluenza virus strain, a preparation method and an application of the inactivated vaccine.
The invention relates to a canine parainfluenza virus strain which has strong toxicity and good immunogenicity.
The invention also relates to a vaccine composition comprising an immunizing amount of the canine parainfluenza virus strain antigen and/or a veterinarily acceptable carrier; wherein the canine parainfluenza virus antigen comprises an inactivated whole virus antigen. The vaccine composition is prepared from a virulent strain, has good immunogenicity, and can provide complete protection for dogs. Compared with the attenuated vaccine in the prior art, the inactivated vaccine has obvious biological safety.
The invention also relates to application of the vaccine composition in preparation of medicines for preventing and/or treating diseases related to canine parainfluenza.
The canine parainfluenza virus strain is an epidemic canine parainfluenza virus wild strain, the vaccine composition prepared from the strain can prevent and/or treat canine parainfluenza epidemic spread, and the vaccine composition containing the strain can enable an animal body to quickly generate antibodies after immunizing animals, has good prevention and control effects on single or mixed infection of the current epidemic canine parainfluenza, and has good biological safety.
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FIG. 1 is a chart of cytopathic and immunofluorescence identification of CPIV HeN0718 and rCPIV on Vero cells; FIG. 1A is a diagram of a blank control Vero cell; fig. 1B is a cytopathy produced by CPIV HeN0718 on Vero cells; FIG. 1C is a cytopathy generated by recombinant rCPIV on Vero cells; FIG. 1D is an immunofluorescence identification plot of blank control Vero cells; FIG. 1E is a chart of immunofluorescence assays generated by CPIV HeN0718 on Vero cells; FIG. 1F is a plot of immunofluorescence generated by rCPIV on Vero cells.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
The term "Canine Parainfluenza virus" (CPIV) belongs to the family paramyxoviridae, the subfamily paramyxoviridae, members of the genus mumps, are nonsegmented single-stranded negative-strand RNA viruses, and cause clinical symptoms including fever, watery nasal discharge, and cough. Pathological changes are characterized by catarrhal rhinitis and bronchitis.
The term "canine parainfluenza virus strain (CPIV strain)" used herein is a canine parainfluenza virus field strain (CPIV field strain), a canine parainfluenza virus virulent strain (CPIV virulent strain), a wild-type CPIV strain, and may be used interchangeably unless otherwise specified.
The term "CDS", which is fully called coding sequence, refers to a sequence in a gene encoding a protein product, which is a DNA sequence corresponding one-to-one to a protein sequence without other sequences than the protein in between, and which completely corresponds to the codon of the protein regardless of sequence changes during mRNA processing or the like.
The invention relates to a canine parainfluenza virus strain, wherein the full-length cDNA of the genome of the canine parainfluenza virus strain is a sequence shown in SEQ ID No.1 or a sequence obtained by replacing CDS in the sequence shown in SEQ ID No.1 with a degenerate sequence thereof.
The invention relates to a canine parainfluenza virus strain, wherein the canine parainfluenza virus strain is a CPIV HeN0718 strain, and the genome full-length cDNA of the canine parainfluenza virus strain CPIV HeN0718 strain is a sequence shown in SEQ ID No. 1.
The isolation of the canine parainfluenza virus strain comprises: sampling, grinding or treating the lesion part of the animal viscera infected with the canine parainfluenza virus, inoculating different grinding liquids or treating liquids into Vero cells for virus separation, stably transmitting to the occurrence of cytopathic effect, and freezing and thawing for virus collection.
Animal sources that infect canine parainfluenza virus strains include, but are not limited to, canine (Canidee) sources, feline (Felidae) sources. Animal samples infected with canine parainfluenza virus strains include, but are not limited to, lung, saliva, ocular secretions, trachea, nasal secretions, lymph nodes, spleen.
The term "vaccine composition," also known as an immunogenic composition, refers to an immunogen-containing preparation, including, for example, whole cells, inactivated or attenuated, live viruses or bacteria, or polysaccharides, or combinations thereof, that are administered to stimulate the humoral and cellular immune response of a recipient to one or more antigens present in the immunogenic composition. Immunization is the process of administering a vaccine composition and stimulating an immune or immunogenic response to an antigen in a host, preferably an animal such as a dog.
The term "immunizing amount" shall be understood as an "immunologically effective amount," also referred to as an immunoprotective amount or an amount effective to produce an immune response, of antigen effective to induce an immune response in a recipient, sufficient to prevent or ameliorate the signs or symptoms of disease, including adverse health effects or complications thereof. The immune response may be sufficient for diagnostic purposes or other testing, or may be suitable for use in preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.
The term "canine parainfluenza virus antigen" refers to any composition containing at least one canine parainfluenza virus antigen form that induces, stimulates or is capable of resisting an immune response against canine parainfluenza virus infection, including but not limited to inactivated, attenuated or subunit antigens. Where the inactivated antigen is inactivated by various means including chemical treatment using, for example, formalin or formaldehyde, beta-propiolactone, ethyleneimine, binary ethyleneimine BEI, thimerosal, or the like, physical treatment (e.g., sonication, irradiation, heat), or any other commonly used means sufficient to prevent replication or growth of the organism, preferably the pathogen is inactivated after collection and optionally subjected to clarification and purification; methods for inactivation are well known to the person skilled in the art, e.g.by treatment with beta-propiolactone (Plana-Duran et al, vet. Microbiol.,1997,55:361-370) or by BEI (US 5587164).
The term "veterinarily acceptable carrier" refers to all ingredients other than the canine parainfluenza virus antigen in the vaccine composition of the present invention, carriers or diluents, preferably adjuvants, that do not stimulate the body and do not hinder the biological activity and properties of the compounds used. The term "adjuvant" may include an alumina gel adjuvant; saponins (saponin), such as Quil A, QS-21(Cambridge Biotech Incorporation, Cambridge MA), GPI-0100(Galenica Pharmaceuticals Incorporation, Birmingham AL); a water-in-oil emulsion; an oil-in-water emulsion; a water-in-oil-in-water emulsion; polymers of acrylic acid or methacrylic acid; maleic anhydride and alkenyl (alkenyl) derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils (isoprenoid oils) resulting from the oligomerization of olefins, such as squalane (squalane) or squalene oil (squalene oil), in particular isobutene or decene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearic acid esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (such as, for example, anhydrous mannitol oleate), of aliphatic diols (glycols), of polyglycerols, of propylene glycol and of oleic acid, of isostearic acid, of ricinoleic acid or of hydroxystearic acid, which are optionally ethoxylated, and also polyoxypropylene-polyoxyethylene block copolymers, in particular the Pluronic products, in particular L121. See The description of The same and The reactive application of adjuvants by Hunter et al (Ed. by DES Stewart-Tull, John Wiley and Sons, New York,1995:51-94) and The description of Vaccine by Todd et al (1997,15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of Vaccine design, the Subunit and adivant propaach (Plenum Press,1995) written by Powell M and Newman M can be used. The term "polymer of acrylic or methacrylic acid" is preferably a crosslinked polymer of acrylic or methacrylic acid, in particular a polyalkenyl ether or polyalcohol crosslinked with a sugar (sugar), these compounds being known under the name Carbomer (Carbopol, trade name Carbopol) (Phameuropa,1996,8 (2)). Those skilled in the art can also see US2909462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are substituted by unsaturated aliphatic hydrocarbon groups (aliphatic radial) having at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups (ethylenically unsaturated groups). The unsaturated groups may themselves contain other substituents, such as methyl. These products are sold under the name carbopol, (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among these, mention may be made of carbopols 974P, 934P and 971P, the most preferred being the use of carbopol 971P. The term "copolymers of maleic anhydride and alkenyl derivative" also contemplates the maleic anhydride and ethylene copolymers ema (monsanto), which are dissolved in water to give an acidic solution, neutralized, preferably to physiological pH, in order to give an adjuvant solution into which the immunogenic, immunogenic or vaccinal composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, the RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A), Avridine lipoamine adjuvant, E.coli heat labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, and the like. Preferably, the adjuvant comprises one or more of an alumina Gel adjuvant, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion, a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl (alkenyl) derivative, a RIBI adjuvant system, a Block co-polymer, SAF-M, a monophosphoryl lipid A, Avridine lipid-amine adjuvant, escherichia coli heat labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide or Gel adjuvant.
The invention also relates to a vaccine composition, wherein the vaccine composition comprises an immunizing amount of the canine parainfluenza virus strain or the inactivated antigen of the culture thereof and a veterinary acceptable carrier.
In one embodiment of the present invention, in the vaccine composition of the present invention, the antigen of canine parainfluenza virus strain is the antigen of canine parainfluenza virus strainInactivated antigen of parainfluenza virus strain CPIV HeN0718 strain or culture thereof, wherein the inactivated antigen content of the canine parainfluenza virus strain CPIV HeN0718 strain or culture thereof is 10 before inactivation4.0-107.0TCID50/ml。
In one embodiment of the present invention, in the vaccine composition of the present invention, the inactivated antigen content of the canine parainfluenza virus strain CPIV HeN0718 strain or its culture is 10 before inactivation5.0-106.0TCID50/ml。
In one embodiment of the present invention, in the vaccine composition of the present invention, the inactivated antigen content of the canine parainfluenza virus strain CPIV HeN0718 strain or its culture is 10 before inactivation6.0TCID50/ml。
As an embodiment of the present invention, the inactivated whole virus antigen is obtained by inactivating a whole virus culture solution obtained by propagating the canine parainfluenza virus strain on cells with an inactivating agent.
As an embodiment of the present invention, the inactivating agent includes, but is not limited to, β -propiolactone BPL, diethyleneimine BEI, formalin, formaldehyde, N-acetylethyleneimine AEI, polyhexamethyleneguanidine hydrochloride PHMG.
In one embodiment of the present invention, in the vaccine composition of the present invention, the culture of canine parainfluenza virus strain is a culture cultured for 1-18 generations.
In one embodiment of the present invention, in the vaccine composition of the present invention, the culture of canine parainfluenza virus strain is a culture cultured for 6-18 generations.
As an embodiment of the present invention, in the vaccine composition of the present invention, the adjuvant comprises: (1) alumino-gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide and Gel adjuvant;
preferably, the saponin is Quil A, QS-21, GPI-0100;
preferably, the emulsion is an SPT emulsion, an MF59 emulsion, or an emulsion formed from an oil in combination with an emulsifier, the emulsion may be based on light liquid paraffin oil, isoprenoid oil resulting from the oligomerization of olefins (such as squalane or squalene oil, oil resulting from the oligomerization of olefins, in particular isobutene or decene), linear alkyl-containing esters of acids or alcohols (more particularly vegetable oil, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate), esters of branched fatty acids or alcohols (in particular isostearate); the emulsifier is a nonionic surfactant (especially esters of polyoxyethylated fatty acids (e.g. oleic acid), sorbitan, mannide (e.g. anhydrous mannitol oleate), aliphatic diols, glycerol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which may be ethoxylated, ethers of fatty alcohols and polyhydric alcohols (e.g. oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers (especially
Figure BDA0001141288530000071
In particular L121));
preferably, the polymer of acrylic acid or methacrylic acid is a crosslinked polymer of acrylic acid or methacrylic acid, in particular a compound carbomer crosslinked with polyalkenyl ethers or polyalcohols of sugars, preferably carbopol 974P, 934P and 971P;
preferably, the copolymer of maleic anhydride and alkenyl derivative is a copolymer EMA of maleic anhydride and ethylene;
preferably, the adjuvant is a GEL a adjuvant;
the concentration of the adjuvant ranges from 5% to 50% V/V, preferably 5% V/V.
As an embodiment of the present invention, the vaccine composition of the present invention further comprises other antigens, wherein the antigens comprise one or more of the following antigens: canine Distemper Virus (CDV) antigen, Canine Adenovirus (Canine Adenoviral) type I, type II antigen, Canine Leptospira interrogans (Leptospira interrogans) antigen, Canine Coronavirus (Canine Coronavir, CCV) antigen, Canine Parvovirus (Canine Parvoviruses, CPV) antigen, Rabies Virus (RV) antigen, Canine Influenza Virus (Canine Inueflu Virus, CIV) antigen, Canine Reovirus (CRV) antigen, Canine Pseudorabies Virus (Pseudoorabes) antigen, Canine Rotavirus (Rotavirus) antigen, Canine herpesvirus (Canine Hervirous, CHV) antigen, Canine Viral Pacosis antigen, Canine Parvovirus (Canine Virus) antigen, Mumps Virus (Mumps Virus) antigen, Mumps Virus.
As a preferred embodiment of the present invention, in the vaccine composition of the present invention, the antigen comprises a group consisting of one or more of the following antigens: canine parvovirus antigen, canine adenovirus antigen, canine leptospira antigen, canine coronavirus antigen, canine parainfluenza virus antigen, rabies virus antigen, canine influenza virus antigen.
The invention also relates to a preparation method of the vaccine composition, which comprises the following steps: and (3) proliferating the canine parainfluenza virus strain on cells, inactivating the canine parainfluenza virus cell sap subjected to amplification culture by using an inactivating agent, and adding a carrier acceptable in veterinary medicine to mix to obtain the canine parainfluenza virus cell sap.
As a preferred embodiment of the present invention, the vaccine composition of the present invention comprises the content of 10 before inactivation4.0-107.0TCID50The inactivated antigen of the/ml canine parainfluenza virus strain CPIV HeN0718 strain with the content of 10 before inactivation4.0-106.0TCID50The inactivated antigen of the canine distemper virus CDVC/HN/001 strain/ml and/or the content of the inactivated antigen is 10 before inactivation6.0-108.0TCID50The inactivated antigen of the canine parvovirus CPV S0425 strain in/ml and the adjuvant of 5% V/VGEL A.
As a preferred embodiment of the inventionThe vaccine composition of the invention comprises the content of 10 before inactivation6.0TCID50The inactivated antigen of the/ml canine parainfluenza virus strain CPIV HeN0718 strain with the content of 10 before inactivation .50TCID50The inactivated antigen of CDV C/HN/001 strain of canine distemper virus of/ml and/or the content of the inactivated antigen is 10 before inactivation7.0TCID50The inactivated antigen of the canine parvovirus CPV S0425 strain in/ml and the adjuvant of 5% V/VGEL A.
In the vaccine composition of the invention, other antigen components generate synergistic effect on inactivated canine parainfluenza virus inactivated antigen, so that the immune dogs can achieve the titer of neutralizing antibodies which can resist the strong virus attack within a shorter time.
The term "preventing and/or treating" when referring to a canine parainfluenza virus infection refers to inhibiting replication of the canine parainfluenza virus, inhibiting transmission of the canine parainfluenza virus, or preventing colonization of the canine parainfluenza virus in its host, as well as alleviating the symptoms of a disease or disorder of canine parainfluenza virus infection. Treatment is considered to be therapeutically effective if the viral load is reduced, the condition is reduced and/or the food intake and/or growth is increased.
The invention also relates to application of the vaccine composition in preparation of a medicament for preventing and treating diseases related to canine parainfluenza virus infection.
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group.
In order that the invention may be more readily understood, reference will now be made to the following examples. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 isolation and identification of canine parainfluenza Virus
Isolating a CPIV wild strain from a clinically canine parainfluenza virus infected animal with clinical symptoms: mental depression, fever, massive mucus effusions from the nasal cavity, opaque secretions, dry cough, and the like, which are taken as criteria for animals to be selected. Taking lung tissue of the infected animal, and grinding the lung tissue by using a tissue homogenizer to obtain tissue homogenate.
Diluting lung tissue homogenate of a sick dog into 10% W/V suspension by using serum-free DMEM culture solution, filtering and sterilizing by using a 0.22-micron filter membrane, inoculating Vero single-layer cells (purchased from Shanghai enzyme-linked detection technology, Inc.) into filtrate according to the 10% W/V ratio for virus separation, wherein cell fusion lesions appear in 24-48 hours, the cell lesions reach more than 80% in about 96 hours, and freeze-thawing virus collection is used as a 1 st generation virus solution; inoculating Vero cells with the virus solution obtained from the 1 st generation by using a synchronous inoculation method, wherein cytopathic effect of cell fusion occurs after 24 hours of inoculation, the cytopathic effect reaches more than 80% after 72-96 hours of inoculation, and freeze-thawing virus collection is used as virus solution of the 2 nd generation; inoculating Vero cells with the virus solution obtained in the 2 nd generation by using a synchronous inoculation method, wherein cytopathic effect of cell fusion occurs after inoculation for 24 hours, the cytopathic effect reaches more than 80% after inoculation for 72-96 hours, and freeze-thawing virus collection is used as virus solution in the 3 rd generation. The 3 rd generation virus liquid is observed by an electron microscope to carry out morphological detection, and the result shows that: viral particles with typical parainfluenza virus characteristics are visible. The indirect immunofluorescence method of the development and characteristic identification of the monoclone antibody of canine parainfluenza virus of Jia Kuhua (Jia Kuhua, Cen Hao, Zhao, Wu Yan Tao, Canine parainfluenza virus [ J ].2009,31(12): 985-: the cytopathic part of the Vero cell inoculated virus formed cell fusion shows specific green fluorescence, which indicates that the specificity is good.
EXAMPLE 2 cloning of canine parainfluenza Virus
1. Sequencing of canine parainfluenza virus whole genome
13 pairs of primers were designed according to the CPIV5 sequence (JQ743318.1) submitted in GenBank, and the amplified fragments partially overlapped with each other and linked to cover the whole genome, and the primer sequences are shown in Table 1.
TABLE 1 Canine parainfluenza Virus Whole genome sequencing primer sequence Listing
Figure BDA0001141288530000101
Figure BDA0001141288530000111
The total RNA of the CPIV virus solution isolated in example 1 was extracted with reference to the instructions of the RNA extraction kit, reverse transcription was performed using the downstream primers P1 to P13, respectively, to prepare viral cDNA, and the whole viral genome was amplified using the obtained cDNA as a template and the designed 13 primer pairs. After the size of the PCR product is identified to be consistent with the expected size through 1% agarose gel electrophoresis, the PCR product is recovered and purified according to the instructions of the gel recovery kit. Cloning the recovered DNA fragment to pEASY-Blunt vector, transforming into E.coli DH5 alpha competent cell, screening positive clone, and sequencing and analyzing, the result is shown in sequence 1.
2. Construction of full-Length infectious clone pCMVTNT-FL
According to analysis of enzyme cutting sites in a canine parainfluenza virus genome sequence 1 and a transcription vector pCMVTNT, a genome is divided into 5 fragments, a target fragment is obtained through an artificial synthesis method and is respectively connected into a cloning vector pEasy-Blunt Simple, then the 5 fragments are sequentially connected into the transcription vector through enzyme cutting and connection, and finally the transcription vector pCMVTNT-FL containing the virus full-length cDNA is obtained. And pCMVTNT-FL was sequenced, and the primer sequences are shown in Table 2. The aligned sequence is completely consistent with the sequence 1.
TABLE 2 sequence Listing of primers for sequencing of full-Length infectious clone pCMVTNT-FL
PV1-F 5’-CGACGCGTACCAAGGGGAAAATGAAGTGG-3’
PV1-R 5’-AGATAGGAGACCATCAGAAGTTG-3’
PV2-F 5’-CGACGCGT ATAGCTAGAATCACCAGCCTG-3’
PV2-R 5’-ACTGTACTGATAGAGCAGCTCTTACG-3’
PV3-F 5’-TCATTCTCGCTCACCAAGACACACTGGTG-3’
PV3-R 5’-TGACATAGCCCTTCAATTCCACCTCTGG-3’
PV4-F 5’-CTAGCTAGCTGGAGGTACCAGACAATTATCC-3’
PV4-R 5’-TGCAGCTGTTCAAGTGTGATGTTAACTC-3’
PV5-F 5’-CTAGCTAGCACCGTACATTGGCTCCAGG-3’
PV5-R 5’-ATAAGAATGCGGCCGCACCAAGGGGAAAACCA-3’
3. Construction of helper plasmids
And (2) artificially synthesizing NP and P gene sequences contained in the sequence 1 to obtain target fragments, respectively cloning the target fragments to a pCI-neo vector, transforming the target fragments into Escherichia coli DH5 alpha competent cells, screening positive clones, carrying out enzyme digestion to identify correct plasmids, namely pCI-NP and pCI-P, and carrying out sequencing analysis. The primer sequences are shown in Table 3.
Dividing the L gene contained in the sequence 1 into L1 and L2 fragments, obtaining a target fragment by an artificial synthesis method, carrying out double enzyme digestion on the obtained L1 fragment by NheI and SalI, recovering the L1 fragment, connecting the L1 fragment with a pCI-neo vector subjected to the same double enzyme digestion, and transforming to obtain the plasmid pCI-L1. The obtained L2 fragment was digested with XhoI and SalI, the L2 fragment was recovered, ligated with the pCI-L1 vector digested with the same restriction enzymes, transformed to obtain plasmid pCI-L12, i.e., pCI-L, and subjected to sequencing analysis. The primer sequences are shown in Table 3.
TABLE 3 sequence Listing of helper plasmid sequencing primers
NP-F 5’-TATTGAATTCGCCACCATGTCATCCGTGCTCAAGGCATATG-3’
NP-R 5’-TATAGTCGACCTAGATGTCGAGATCGCCCAGTG-3’
P-F 5’-TATTGAATTCGCCACCATGGATCCCACTGATTTGAGC-3’
P-R 5’-TATAGTCGACTCAGATTGTACTGCGGATGATTGC-3’
L1-F 5’-TATTGCTAGCGCCACCATGGCTGGGTCTCGGGAGATA-3’
L1-R 5’-TATAGTCGACAGAGTATCTCCGAATGCCCAG-3’
L2-F 5’-TCCGTGTACCGTACATTGGCTCCAG-3’
L2-R 5’-TATAGTCGACTTAGATTTCCTCGCCATCGATC-3’
4. Rescue of infectious clones
BHK cells were cultured at 8X 105The concentration of each well was inoculated in 6-well cell plates, when 70% -80% monolayer was grown overnight, the supernatant medium was discarded, and the full-length cDNA clone pCMVTNT-FL of the recombinant virus genome and the helper plasmids pCI-NP, pCI-P and pCI-L were CO-transfected into BHK cells at 1. mu.g, 1. mu.g and 0.1. mu.g, 5% CO, respectively, using FuGENE 6 transfection reagent2Culturing at 37 ℃, and carrying out specific operation according to the kit instruction. Within 14-16 h after transfection, the cell plate is sealed in a water bath at 43 ℃ for 3h with 5% CO2After culturing at 37 ℃ for 48 hours, 8X 10 cells were added per well of transfected cells5Vero cells. Culture was continued until the appearance of a typical canine parainfluenza cell lesion (fig. 1C). When the pathological changes reach more than 80 percent, the recombinant viruses are collected by a repeated freeze-thaw method and are stored at minus 80 ℃ as seed viruses. The rescued virus was named rciv, and a typical canine parainfluenza cytopathy developed on Vero using CPIV HeN0718 strain as a positive control is shown in figure 1B. FIG. 1A is a graph of blank Vero cells.
And then, after infecting Vero cells with the infectious clone rCPIV and the original parent virus CPIV for 48 hours, carrying out virus specificity identification by adopting an indirect immunofluorescence method, wherein the results are respectively shown in figures 1E and 1F, and figures 1E and 1F show that: the cytopathic part of the cell formed by rCPIV infecting Vero cells into cell fusion shows specific green fluorescence, and has no change compared with the original parent virus CPIV. FIG. 1D is an immunofluorescence plot of healthy cells of a blank control group.
Example 3 pathogenicity assay for canine parainfluenza Virus
15 CPIV antigen-antibody negative 2-3 month-old healthy dogs were selected and randomly divided into 3 groups and 5 dogs, and group 1 was inoculated with 2ml of CPIV virus solution (virus content: 10) isolated in example 1 by nasal inoculation5.0TCID50Perml), group 2 nasal inoculation of Virus 2ml of rCPIV virus solution cloned in example 2 (virus content 10)5.0TCID50Ml), group 3 was used as a blank control group, without toxicity control, and was kept separately. And observing for 14 days, wherein clinical characteristics such as spirit, appetite, body temperature, nasal discharge, cough, limb movement coordination and the like of the experimental dog are mainly observed. The results are shown in Table 4.
TABLE 4 pathogenicity test results for canine parainfluenza viruses
Group of Animal numbering Inoculation dose Number of onset of disease
1 1-5 2 ml/piece 5/5
2 6-10 2 ml/piece 5/5
3 10-15 2ml of physiological saline 0/5
As can be seen from Table 4: the CPIV isolated in example 1 and the rCPIV cloned in example 2 both caused morbidity in experimental animals and were consistent in pathogenicity.
Through the comparison of the CPIV isolated strain and the cloned strain in tests such as cytopathic effect, immunofluorescence, animal pathogenicity, gene sequencing and the like, the CPIV isolated strain and the cloned strain have the same phenotype, can be regarded as the same virus strain and are named as HeN0718 strains.
Example 4 Canine parainfluenza Virus HeN0718 strain virulence stability test
The CPIV HeN0718 strain is continuously and naturally passaged on Vero cells, and virus solutions of P1 generation, P5 generation, P10 generation, P15 generation and P18 generation are respectively taken for animal pathogenicity test. Selecting 30 CPIV antigen-antibody negative 2-3 month-old healthy dogs, randomly dividing into 6 groups and 5 dogs, inoculating viruses of P1 generation, P5 generation, P10 generation, P15 generation and P18 generation in 4-8 groups through nasal cavity respectively (the virus content is 10)5.0TCID50Ml), group 9 was used as a blank control group, without toxicity control, and was kept separately. And observing for 14 days, wherein clinical characteristics such as spirit, appetite, body temperature, nasal discharge, cough, limb movement coordination and the like of the experimental dog are mainly observed. The results are shown in Table 5.
TABLE 5 pathogenicity test results of canine parainfluenza virus HeN0718 strains of different generations
Figure BDA0001141288530000141
Figure BDA0001141288530000151
As can be seen from Table 5, the canine parainfluenza virus HeN0718 strains of different generations can be used for testing animal morbidity, and the pathogenicity is consistent.
The canine parainfluenza virus HeN0718 strain of the invention has stable virulence and no virulence attenuation in the cell culture process.
Example 5 preparation of inactivated vaccine of canine parainfluenza virus HeN0718 strain
Inoculating a CPIV HeN0718 strain culture to Vero cells, inoculating the Vero cell culture which forms a monolayer according to the MOI of 0.01, culturing at 37 ℃, harvesting a cell culture solution containing the virus when the cytopathic effect reaches 80%, freezing and thawing for 2 times, collecting the virus, and measuring the toxic value. Adding BPL (beta-propiolactone) with the final concentration of 0.025 percent for inactivation, and performing sterile inspection, mycoplasma inspection and exogenous virus inspection on the inactivated canine parainfluenza virus according to a method of Chinese veterinary pharmacopoeia (China veterinary Committee, three 2010 editions, China agricultural Press, 2010), wherein the results show that: after the CPIV HeN0718 strain is inactivated, the CPIV HeN0718 strain is not polluted by bacteria and mould, is not infected by mycoplasma and exogenous viruses, and has good purity.
And (3) fully and uniformly mixing the inactivated virus solution with Gel A adjuvant purchased from Seppic company to obtain the inactivated vaccines 1, 2, 3 and 4. The specific ratio is shown in Table 6.
TABLE 6 CPIV HeN0718 strain inactivated vaccine ratio
Name (R) Pre-inactivation Virus content (TCID)50/ml) Gel A adjuvant content (V/V%)
Vaccine 1 104.0 5％
Vaccine 2 105.0 5％
Vaccine 3 106.0 5％
Vaccine 4 107.0 5％
Example 6 safety test of inactivated vaccine against canine parainfluenza virus HeN0718 strain
1. Safety test for one single dose vaccination
25 healthy susceptible dogs of about 50 days old are randomly divided into 5 groups and 5 dogs per group, wherein the 10 th group and the 13 th group are respectively inoculated with 1ml of inactivated vaccine 1, 2, 3 and 4 subcutaneously, and the 14 th group is injected with 1ml of normal saline subcutaneously to be used as a blank control. Feeding under the same condition, observing for 14 days, and observing whether the appetite, spirit, body temperature and eyes and nose of the dog are normal or not, and whether adverse reactions occur on the injection part and the whole body or not every day. Results (see table 7): after the susceptible dogs are inoculated by one single dose, the dogs have good mental state, normal diet, normal body temperature and no generation of eye and nose secretion, no adverse reaction is caused on injection parts and the whole body, and all dogs are healthy and alive.
TABLE 7 safety test results of one-time single-dose vaccination of dogs with inactivated vaccine
Figure BDA0001141288530000161
2. Safety test for single dose repeat inoculations
25 healthy susceptible dogs of about 50 days old are adopted and randomly divided into 5 groups and 5 groups, wherein the 15 th group to the 18 th group are respectively inoculated with 1ml of inactivated vaccine 1, 2, 3 and 4 for the first time through subcutaneous inoculation, 1ml of inactivated vaccine is boosted at the 14 th day after the first immunization, and the 19 th group is injected with 1ml of physiological saline for subcutaneous injection to serve as a blank control. Feeding under the same condition, observing for 14 days, and observing whether the appetite, spirit, body temperature and eyes and nose of the dog are normal or not, and whether adverse reactions occur on the injection part and the whole body or not every day. Results (see table 8): after the susceptible dogs are repeatedly inoculated with single dose, the dogs have good mental state, normal diet and normal body temperature, no adverse reaction is caused on injection parts and the whole body, and all dogs are healthy and alive.
TABLE 8 safety test results of inactivated vaccine against single dose repeat vaccination of dogs
Figure BDA0001141288530000162
Figure BDA0001141288530000171
3. Safety test for one-time overdose inoculation
25 healthy susceptible dogs of about 50 days old are randomly divided into 5 groups and 5 dogs per group, wherein the 20 th group to the 23 th group are respectively inoculated with 4ml of inactivated vaccine 1, 2, 3 and 4 subcutaneously, and the 24 th group is injected with 4ml of normal saline subcutaneously to serve as a blank control. Feeding under the same condition, observing for 14 days, and observing whether the appetite, spirit, body temperature and eyes and nose of the dog are normal or not, and whether adverse reactions occur on the injection part and the whole body or not every day. Results (see table 9): after the susceptible dogs are inoculated with the overdose vaccine for one time, the dogs have good mental state, normal diet and normal body temperature, no adverse reaction is caused on injection parts and the whole body, and all dogs are healthy and alive.
TABLE 9 safety test results of one-time overdose vaccination of dogs with inactivated vaccine
Figure BDA0001141288530000172
The above results show that: the inactivated vaccines 1, 2, 3 and 4 prepared by the invention are safe for immunizing dogs.
Example 7 immunogenicity test of inactivated vaccine of canine parainfluenza virus HeN0718 strain
25 healthy susceptible dogs of about 50 days old are randomly divided into 5 groups and 5 dogs per group, wherein the 25 th group to the 28 th group are respectively inoculated with 1ml of inactivated vaccine 1, 2, 3 and 4 subcutaneously, and the 29 th group is injected with 1ml of normal saline subcutaneously to be used as a blank control. The dogs are bred under the same condition, and the dogs are observed for 14 days to have normal appetite, spirit, body temperature and eyes and noses, and have no adverse reaction on the injection part and the whole body.
On day 21 after immunization, 2ml of a virus solution (virus content: 10) of CPIV HeN0718 strain was used5.0TCID50And/ml) attack test dogs, after attacking, whether the dogs are normal in appetite, spirit, body temperature and eyes and noses and whether adverse reactions occur at injection parts and the whole body are observed every day, and the attacking protection rate is calculated, and the results are shown in table 10.
TABLE 10 results of immunogenicity test of inactivated vaccine
Figure BDA0001141288530000181
As can be seen from table 10: the inactivated vaccines 1, 2, 3 and 4 are used for immunizing healthy susceptible dogs of about 50 days old respectively, each inactivated vaccine is 1ml, and after 21 days of immunization, the canine parainfluenza virus HeN0718 strain is used for virus challenge, and the protection rates of the vaccines 1, 2, 3 and 4 to the dogs are all 100%, which indicates that the inactivated vaccines 1, 2, 3 and 4 have good protection effects.
Example 8 preparation of Canine distemper antigen
The Canine Distemper Virus C/HN/001 strain (Canine Distemper Virus, strain C/HN/001 is preserved in China center for type culture collection with the preservation number of CCTCC NO. V201604, the preservation address of university of Wuhan and Wuhan, China, the preservation date of 2016, 3 and 29) is amplified on a monoclonal Cell line Vero/DogSLAM Cell line Vero/DongSLAM-1F5 strain (Vero/DogSLAM Cell line, strain Vero/DongSLAM-1F5 is preserved in China center for type culture collection with the preservation number of CCTCC NO. C201611, the preservation address of university of Wuhan and Wuhan, the preservation date of 2016, 03 and 29), Virus liquid is harvested, and after 2 times of Virus collection and freeze thawing, the Virus price is determined. Adding BPL (beta-propiolactone) with the final concentration of 0.025 percent for inactivation, and performing sterility test, mycoplasma test and exogenous virus test on CDV C/HN/001 according to a method of Chinese veterinary pharmacopoeia (China veterinary Committee, three parts of 2010 edition, China agricultural Press, 2010), wherein the results show that: after the CDV C/HN/001 strain is inactivated, the strain is not polluted by bacteria and mould, is not infected by mycoplasma and exogenous viruses, and has good purity.
Example 9 preparation of Canine parvoVirus antigen
Canine Parvovirus S0425 Strain (Canine Parvovirus, Strain S0425 is preserved in China center for type culture Collection with the preservation number of CCTCC NO.V201634, the preservation address of university of Wuhan, China, the preservation date of 2016, 6, 14) is amplified on F81 cells, virus liquid is harvested, and after 2 times of freeze thawing, virus is harvested, and the virus price is determined. Adding BPL (beta-propiolactone) with the final concentration of 0.025 percent for inactivation, and performing sterility test, mycoplasma test and exogenous virus test on CPV according to a method of Chinese veterinary pharmacopoeia (China veterinary Committee, three 2010 editions, China agricultural Press, 2010), wherein the results show that: after the CPV S0425 strain is inactivated, the strain is not polluted by bacteria and mould, is not infected by mycoplasma and exogenous viruses, and has good purity.
EXAMPLE 10 preparation of vaccine compositions containing canine parainfluenza virus antigens
The CPIV HeN0718 strain antigen prepared in example 5, the CDV C/HN/001 strain antigen prepared in example 8, the CPV S0425 strain antigen prepared in example 9, and the GEL A adjuvant from Seppic were prepared according to the composition and content shown in Table 11.
TABLE 11 vaccine composition content ratio containing canine parainfluenza virus antigen
Figure BDA0001141288530000191
EXAMPLE 11 use of vaccine compositions containing canine parainfluenza virus antigens
1. Safety testing of vaccine compositions containing canine parainfluenza virus antigens
15 healthy beagle dogs of 2-3 months of age, namely CPIV, CDV antigen, antibody negative, CPV antigen negative and antibody HI less than or equal to 1: 4, are selected and randomly divided into 3 groups and 5 groups, the vaccines prepared in the example 10 are immunized respectively in 5-7 groups, 10 doses of the vaccines are injected into each beagle dog, and the observation is carried out for 14 days. As a result: the immunized beagle dogs are normal in spirit and diet; the body temperature has no obvious change and is within the normal range of 38.5-39.5 ℃; no swelling and necrosis appear at the inoculation part, and no systemic adverse reaction occurs. Shows that: the safety tests of the vaccines 5-7 are all qualified.
2. Immunogenicity testing of vaccine compositions containing canine parainfluenza virus antigens
55 healthy beagle dogs with the age of 2-3 months, namely CPIV, CDV antigen, antibody negative, CPV antigen negative and antibody HI not more than 1: 4 are selected and randomly divided into 11 groups, 5 groups, 30-31 groups, 32-33 groups, 6 groups, 34-35 groups and 36 groups, 7 groups and 1 ml. And the 37 th group, the 38 th group and the 39 th group are used as challenge controls, the 40 th group is used as a blank control, and the immunization is not carried out and the isolation feeding is carried out. Blood is collected before immunization and 14 days and 21 days after immunization respectively, and HI antibody titer is detected (the HI antibody titer is detected according to invar, Liujing, animal virology, 2 nd edition, Beijing, science Press, 1997: 204-. After 21 days of immunization, 2ml of virus solution (with a virus content of 10) of canine parainfluenza virus HeN0718 strain was nasally challenged to all the dogs in groups 30, 32, 34 and 375.0TCID50Ml), observed for 14 days. The results are shown in Table 12.
TABLE 12 animal test results after CPIV challenge with vaccine compositions containing CPIV antigen
Figure BDA0001141288530000201
As can be seen from table 12: (1) the antibody titer after immunization, namely the vaccine 5-7 can generate higher CPIV SN antibody titer after immunizing dogs, which shows that the SN antibody titer of the CPIV is not influenced when the inactivated canine parainfluenza virus antigen is mixed with canine distemper virus and canine parvovirus attenuated antigen for use; (2) the virus challenge protection is carried out, after the vaccine is immunized by 5-7, the typical symptoms of the canine parainfluenza virus disease do not appear when the vaccine is challenged by a CPIV virulent strain, and the protection rate is 100 percent.
The data in tables 10 and 12 taken in combination with groups 27, 30, 32 and 34 show that CPIV inactivated antigen at the same level (10)6.0TCID50/ml), a single vaccine containing only CPIV inactivated antigen takes longer than a co-vaccine containing CPIV inactivated antigen (also containing CPV and/or CDV inactivated antigen) to produce the same neutralizing antibody titer; alternatively, at the same time after immunization, a single shoot containing only CPIV inactivated antigen produced a lower titer of neutralizing antibodies than a co-shoot containing CPIV inactivated antigen (which also contains CPV and/or CDV inactivated antigen). That is, the presence of other antigenic components promotes the immunogenic efficacy of CPIV HeN718 to inactivate the antigen, resulting in a synergistic effect.
After 21 days of immunization, all dogs in groups 31, 35 and 38 were challenged with 6ml of canine parvovirus strain S0425 virus solution (virus content 10) by oral route7.0TCID50Ml), observed for 14 days. The results are shown in Table 13.
TABLE 13 animal test results after CPV challenge for vaccine compositions containing CPIV antigen
Figure BDA0001141288530000211
As can be seen from table 13: (1) the antibody titer after immunization, namely the vaccine 5 and the vaccine 7 can generate higher CPV HI antibody titer after immunizing dogs, which shows that the CPV HI antibody titer is not influenced when the canine parvovirus inactivated antigen is mixed with canine parainfluenza virus and canine distemper virus antigens for use; (2) the virus challenge protection is carried out, after the vaccines 5 and 7 are immunized, the virus challenge with CPV epidemic virulent strains does not have the symptom of canine parvovirus disease, and the protection rate is 100 percent.
After 21 days of immunization, all dogs in groups 33, 36 and 39 are attacked by CDV C/HN/001 strain virus solution (the virus content is 10) through nasal drip of 1ml and intraperitoneal injection of 2ml5.0TCID50Ml), observed for 14 days, and the results are shown in Table 14.
TABLE 14 animal test results after CDV challenge with vaccine compositions containing CPIV antigen
Figure BDA0001141288530000212
As can be seen from table 14: (1) the antibody titer after immunization, namely the vaccine 6 and the vaccine 7 can generate higher CDV SN antibody titer after immunizing dogs, which shows that the CDV SN antibody titer is not influenced when the canine distemper virus inactivated antigen is mixed with canine parainfluenza virus and canine parvovirus antigen for use; (2) the virus challenge protection is carried out, after the vaccines 6 and 7 are immunized, the virus challenge with CDV epidemic virulent strains does not have the symptoms of canine distemper virus diseases, and the protection rate is 100 percent.
In summary, the following steps: after the vaccine composition containing the canine parainfluenza virus antigen is immunized, typical clinical symptoms of the canine parainfluenza virus diseases such as appetite reduction, body temperature rise, rhinorrhea, vomiting, cough, spasm, convulsion and the like do not appear, the vaccine composition does not have immune interference effect on other antigens contained in the vaccine composition, and the other antigens contained in the vaccine composition can also generate synergistic effect on the canine parainfluenza virus antigen.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> Puleco bioengineering GmbH
<120> canine parainfluenza virus strain and application thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 15246
<212> DNA
<213> Canine parainfluenza Virus
<400> 1
accaagggga aaatgaagtg gtgacccaaa tcatcgaaga ccctcgagat tacataggtc 60
cggaacctat ggccttcgtg accgacctcg agtcagagta gttcaataag gacctatcaa 120
gtttgggcaa ttttttgtct ccgacacaaa aatgtcatcc gtgctcaagg catatgagcg 180
attcacgctc actcaagaac tgcaagatca gagtgaggaa ggtacaatcc cacctacaac 240
actaaaaccg ataatcaggg tatttatact aacctctaat aacccagagc taagatcccg 300
gcttcttcta ttctgcctac ggattgttct cagtaatggc gcaagggatt ctcatcgctt 360
tggagcatta cttacaatgt tttcactacc atcagccaca atgctcaatc atgtcaaatt 420
agctgaccag tcaccagaag ccgatatcga aagggtagag atcgatggtt ttgaggaggg 480
atcattccgc ttaatcccca atgctcgttc aggcatgagc cgtggagaga tcaatgccta 540
tgctgcactt gcagaagatc tacctgacac actaaaccat gaaacacctt ttgttgattc 600
cgaagtcgag gggactgcat gggatgagat tgagactttc ttagatatgt gttacagtgt 660
cttaatgcag gcatggatag tgacttgcaa gtgtatgact gcgccagacc aacctgctgc 720
ctctattgag aaacgcctgc aaaaatatcg tcagcaaggc aggatcaacc caagatatct 780
cctgcaaccg gaggctcgac gaataatcca gaatgtaatc cggaagggga tggtggtcag 840
acatttcctc accttcgaac tgcagcttgc ccgagcacaa agccttgtat caaataggta 900
ttatgctatg gtaggggatg ttgggaagta tatagagaat tgtggaatgg gaggcttctt 960
tttgacacta aaatatgcat taggaaccag atggcccaca cttgccctag ctgcattttc 1020
aggagagcta acaaagctaa agtccctcat ggcattatac cagacccttg gtgagcaggc 1080
ccgatacttg gccctattgg agtcaccaca tttgatggat tttgctgcag caaactaccc 1140
actgctatac agctatgcta tgggaatagg ctatgtgtta gatgtcaaca tgaggaacta 1200
tgctttctcc agatcataca tgaataagac atatttccaa ttaggaatgg aaactgcaag 1260
aaaacaacag ggtgcagttg acatgaggat ggcagaagat ctcggtctaa ctcaagccga 1320
acgcaccgag atggcaaata cacttgccaa attgaccaca gcaaatcgag gggcagacac 1380
caggggagga gtcaaccctt tctcatctgt caccgggaca actcaggtgc ccgctgcggt 1440
aacaggtgac acattcgaga gctacatggt cgcggatcga ctgaggcaga gatatgctga 1500
tgcaagcacc cacgatgatg agatgccacc actggaagag gaggaagaag acgatacatc 1560
tgcaggtcca cgcactggac caactctcga acaagtggcc cttgacatcc agaacacagc 1620
agttggagct ccaatccata cagatgactt gaatgccgca ctgggcgatc tcgacatcta 1680
gacaattcag atctcaatcc aaaatcgaca cacccaagta accagctaga tggaaccaca 1740
gtggactcca caaggttcct gcccactaca cgcttttaag aaaaaaatag gcccggacgg 1800
gttagtaaca agtgatcgcc gatgtcaata acacaatcca caatctacaa tggatcccac 1860
tgatttgagc ttctccccag atgagatcaa caagctcata gagacaggcc tgaacactgt 1920
ggagtatttt acttcccagc aagtcacagg aacatcctct cttggaaaga atacaatacc 1980
accaggggtt acaggactac taaccaatgc tgcagaggca aagatccaag agtcagccaa 2040
ccatcagaag gttccagttg atgggggtgc aaaatcaaag aaaccgcgac caaaaattgc 2100
cattgtgcca gcagatgaca aaacggtgcc cgggaagccg atcccaaacc ctctattagg 2160
tctggactcc accccaagca cccaaactgt gcttgatcta agtgggaaaa cattaccatc 2220
aggatcctat aaaggggtta agcttgcgaa atttggaaaa gaaaatctga tgacacggtt 2280
catcgaggaa cccagagaga atcctatcgc aaccagttcc ccaaccgatt ttaagagggg 2340
cagggatacc ggagggttcc atagaaggga gtactcaatt ggatgggtgg gagatgaagt 2400
caaggtcact gagtggtgca atccatcctg ttctccaatc accgctgcag caaggcgatt 2460
tgaatgcact tgtcaccagt gtccagtcac ttgctctgaa tgtgaacgag atacttaata 2520
cagtgagaaa tctggactct cggatgaatc aactggagac aaaagtagat cgcattctct 2580
catctcagtc tctaatccag accatcaaga atgacatagt tggactcaaa gcagggatgg 2640
ctactttaga aggaatgatt acaactgtga aaattatgga cccgggagtt cccagcaatg 2700
ttactgtgga agatgtgcgc aagaaattaa gtaaccatgc tgttgttata ccagaatcat 2760
tcaatgatag tttcttgact caatctgaag atgtaatttc acttgatgag ttggcccgac 2820
caactgcaac aagtgttaag aagattgtca ggaaggttcc tcctcagaag gatctgactg 2880
gattgaagat cacactagag caattggcaa aggattgcat cagtaaaccg aagatgaggg 2940
aagagtatct ccttaaaatc aaccaggctt ccagtgaggc ccagctactt gacctcaaga 3000
aagcaatcat ccgcagtaca atctgatcta gaaacaccca attacaccac actggcatga 3060
cactgcacca accctgaggg ctttagaaaa aacgatcgat aataaataag cccgaacact 3120
atacaccacc cgaggcagcc atgccatcca tcagcatccc tgcagaccct accaatccac 3180
gtcaatcaat aaaagcgttc ccaattgtga ttaacagtaa tgggggtgag aaaggccgct 3240
tagttaaaca actacgcaca acctacttga atgacctaga tactcatgag ccactggtga 3300
cgttcgtaaa tacttatgga ttcatctacg aacagactcg gggaaatacc attgtcggag 3360
aggatcaaca tgggaagaaa agagaggctg tgactgctgc aatggttact cttggatgtg 3420
ggcctaatct accatcatta gggaatgtcc tgggacaact aagtgaattc caggtcattg 3480
ttagaaagac atccagcaaa gcagaagaga tgatctttga aattgttaag tatccgagaa 3540
tatttaggag tcatacatta atccagaaag gactagtctg tgtctccgca gaaaaatttg 3600
ttaagtcacc agggaaagta caatctggaa tggactatct cttcattccg acatttttgt 3660
cagtgactta ctgtccagct gcaatcaaat ttcaggtacc tggccctatg ttaaagatga 3720
gatcaagata cactcagagc ttacaacttg aactaatgat aagaatcctg tgtaagcccg 3780
attcgccact tatgaaggtc cacatccctg acaaggaagg aagaggatgt cttgtatcag 3840
tttggttgca tgtatgcaac atctacaaat caggaaacaa gaatggcagt gagtggcagg 3900
aatactggat gagaaagtgt gctaacatgc aacttgaagt gtcgattgca gacatgtggg 3960
gaccaaccat cataattcat gccaggggtc acattcccaa aagtgctaag ttgtttttcg 4020
gaaagggtgg atggagctgc catccacttc atgaagttgt tccaagtgtc accaaaacac 4080
tatggtctgt aggctgtgag attacaaagg ctaaggcaat catacaagag agtagcatct 4140
ctcttctcgt ggagactact gacatcataa gtccaaaagt caaaatttca tctaagcatc 4200
gtcgctttgg gaaatcaaat tggggtctgt tcaggaaaac cagatcacta cccaacctga 4260
cgaagctgga atgactgacc tctaatcgag attacaccac ctcaaactat agatgggtgg 4320
tacctaagtg atcaatcttg taagtactga tcgtaggcta caacacatta atattattcg 4380
ggttagatag ctcaattagc tctttattaa taataacact actattccaa tagctagaat 4440
caccagcctg atttatctcc aaaatgattc aaagaaaaca agtcatatta agactatctt 4500
aagcacgaac ccatatcgtc cttcaaatca tgggtactag aattcaactt ctgatggtct 4560
cctatctatt ggcaggaaca ggcagccttg atccagcagc cctcatgcaa atcggtgtca 4620
ttccaacaaa tgtccggcaa cttatgtatt atactgaggc ttcatcagca ttcattgttg 4680
tgaagttaat gcctacaatt gactcgccga ttagtgggtg taatataaca tccatttcaa 4740
gctataatgc aacaatgaca aaacttctac agccgatcgg tgagaattta gagacgatta 4800
ggtaccagtt gattccaact cggaggagac gccggtttgt aggggtggtg attggattgg 4860
ccgcattagg agtagctact gcagcacagg ttactgccgc agtagcacta gtaaaggcga 4920
ataaaaatgc tgcggctata ctcaatctca aaaatgcaat ccaaaaaaca aatgcagcag 4980
ttgcagatgt ggttcaggcc acacaatcac taggaacggc agttcaagca gttcaagatc 5040
acataaatag tgtggtaagt ccagcaatta cagcagccaa ttgcaaagcc caagatgcta 5100
tcattggctc aattctcaat ctttatttga ccgagttgac aactatcttc cataatcaaa 5160
ttacaaaccc cgcattgagt cctattacaa ttcaagcttt aaggatccta ctagggagca 5220
ccttgccgac cgtggtcaga aaatctttca atacccagat aagtgcagct gagcttctct 5280
catcagggtt attgacaggc cagattgtgg gattagattt gacctacatg cagatggtca 5340
taaaaattga gctgccaact ttaactgtac aacctgcaac tcagatcata gatctagtca 5400
ccatttctgc attcattaac aatcgagaag ttatggccca attaccaaca cgtgttattg 5460
tgactggcag cttgatccaa gcctatcccg catcgcaatg tactatcacc cccaacactg 5520
tgtattgtag gtataatgat gcccaagtac tctcagataa tacgatggct tgcctccaag 5580
gtaacttgac aagatgcacc ttctctccgg tggttgggag ctttcttact cgattcgtgc 5640
tgtttgatgg aatagtttat gcaaattgca ggtcgatgtt gtgcaagtgc atgcagcctg 5700
ctgctgttat cctacagcca agttcatccc ctgtaactgt cattgacatg cacaagtgtg 5760
tgagtctgca gcttgacaat ctcagattca ccatcactca attggccaat ataacctaca 5820
atagcaccat caagcttgaa acatctcaga tcttgcctat tgatccgttg gatatatccc 5880
agaatctagc tgcggtgaat aagagtctaa gtgatgcact acaacactta gcacaaagtg 5940
acacatacct ttctgcaatc acatcagcta cgactacaag tgtattatcc ataatagcaa 6000
tctgtcttgg atcgttaggt ttaatattaa tagtcttgat cagtgtagtt gtgtggaagt 6060
tattgaccat tgtcgctgct aatcgaaata gaatggagaa ttttgtttat cataattcag 6120
catcccacca ctcacggtct gatctcagtg agaaaaatca acctgcaact cttggaacaa 6180
gataagacag tcatctatta ataattttaa agaaaaaaac gataggaccg aaactagtat 6240
tgaaagaacc gtctcggtca atccaggcaa tcaagccgac accgtctcgg aaagctcaaa 6300
tcatgctgcc cgatccggaa gatccggaga gcaaaaaaac tacaaggaga acaggaaacc 6360
caatcatccg ctttccattc accctccctc tatttgtaaa cttcactgtc ccaactccaa 6420
gacacccact gtcccaacac ttgccatagg ccatctacta tatcatctct cctgccatac 6480
ttcctattca catcatatct attttaaaga aaacataggc ccgaacacta atcgtgccgg 6540
cagtgccact gcacacacaa cactacacgt acaatacact acaatggttg cagaagatgc 6600
ccctgttagg ggcacttgcc gagtattatt tcgaacaata actttaattt ttctatgcac 6660
actactagca ttaagcatct ctatccttta tgagagttta ataacccaag agcaaataat 6720
gagccacgca ggctcaactg gatctaattc tcgattagga agtatcattg atcttcttaa 6780
taatattctc tctgtcgcaa atcagattat atataactct gcagtcgctc tacctctaca 6840
attggacact cttgaatcaa cactccttac agccattaag tctcttcaaa caagtgacaa 6900
gctagaacag aactgctcat ggggtgctgc actgattaat gataatagat acattaatgg 6960
catcaatcag ttttatttct caattgctga gggtcgcaat ctggcacttg gcccacttct 7020
taatatacct agtttcattc caactgccac gacaccagag ggctgcacca ggatcccatc 7080
attctcgctc accaagacac actggtgtta tacacacaat gttatcctga atggatgcca 7140
ggatcatgta tcctcaaatc aatttgtttc catgggaatc attgagccca cttctgccgg 7200
gtttccatcc tttcgaaccc taaagactct atatctcagc gatggggtca atcgtaagag 7260
ctgctctatc agtacagttc cggggggttg tatgatgtat tgtttcgtct ctactcaacc 7320
agagagggat gactaccttt ctaccgctcc tccagaacaa cgaattatta taatgtacta 7380
taatgataca atcgtggagc gcataattaa tccacccgga gtactagatg tatgggcaac 7440
attgaaccca ggaacaggaa gcggggtata ttatttaggt tgggtactct ttccaatata 7500
tggcggcgtg attaaaaaga cgagtttatg gaataatcaa gcaaataaat actttatccc 7560
ccagatggtt gctgctctct gctcacaaaa ccaggcaact caagtcaaaa atgctaagtc 7620
atcatactat agcagctggc tcggcaatcg aatgattcag tctggaatcc tggcatgtcc 7680
tcttcaacag gatctaacta atgagtgttt agttctgccc ttttctaatg atcaggtgct 7740
tatgggtgct gaagggagat tatacatgta tggtgactcg gtgtattact accaaagaag 7800
caatagttgg tggcctatga ccatgctgta taaggtaacc ataacgttca ctaatggtca 7860
gccatctgct atatcagctc agaatgtgcc cacacagcag gtccctagac ctgggacaag 7920
aaactgctct gcaacaaata gatgtcccgg tttttgcttg aaaggagtgt atgctgatgc 7980
ctggttactg accaacccct cgtctaccag cacatttgga tcggaagcaa ccttcactgg 8040
ctcttatctc aacgcggcaa ctcagcgtat caatccgacg atgtatatcg cgaacaacac 8100
acagatcata agctcacagc aatttggatc aagcggtcaa gaagcagcat atggtcacac 8160
aacttgtttt agggacacag gctctgttat ggtatactgt atctatatca ttgaactgtc 8220
atcatccctc ttaggacaat ttcagatagt tccatttatc cgtcaggtga cactatccta 8280
aaggcaaaag catccaggtc tgacccagca aatcaaggca ttataccaga ccatgaaatg 8340
tataccaaac attattaaca ctaatgacac acaaaaatgg ttttaagaaa aaccaagaga 8400
acaataggcc agaatggctg ggtctcggga gatattactc cctgaagtcc atctcaattc 8460
gccaattgta aagcataagc tatactatta cattctactt ggaaacctcc caaatgagat 8520
cgacattgac gatttaggtc cattacataa tcaaaattgg aatcagatag cacatgaaga 8580
gtctaactta gcccaacgct tggtaaatgt aagaaatttt ctaattactc acatccctga 8640
tcttagaaag ggccattggc aagagtatgt caatgtaata ctgtggccgc gaattcttcc 8700
cttgattccg gattttaaaa tcaatgacca attacctcta ctcaaaaatt gggacaagct 8760
agttaaagaa tcatgttcag taatcaatgc gggtacttcc cagtgtattc agaatctcag 8820
ctatggactg acaggtcgtg ggaacctctt tacacgatca cgtgaactct ctggtgaccg 8880
cagggatatt gatcttaaga cggttgtggc ggcatggcat gactcagact ggaaaagaat 8940
aagtgatttt tggattatga tcaaattcca gatgagacaa ttaattgtta ggcaaacaga 9000
tcataatgat cctgatttaa tcacatatat cgaaaacaga gaaggcataa tcatcataac 9060
tcctgaactg gtagcattat ttaacactga gaatcataca ctaacatata tgacctttga 9120
aattgtactg atggtttcag atatgtacga aggtcgtcac aacattttat cactatgcac 9180
agttagcact tacctgaatc ccctgaagaa aagaataaca tatttattga gccttgtaga 9240
taacttggct tttcagatag gtgatgctgt atacaacata attgctttgc tagaatcctt 9300
tgtatatgca cagttgcaaa tgtcagatcc catcccagaa ctcagaggac aattccatgc 9360
attcgtatgt tctgagattc ttgatgcact aaggggaact aatagcttca cccaagatga 9420
atcaagaaat gtgacaacca atttgatatc cccattccaa gatctgaccc cagatcttac 9480
ggctgaattg ctctgtataa tgaggctttg gggacacccc atgctcactg ccagtcaagc 9540
tgcgggaaag gtacgcgagt ctatgtgtgc tggaaaggtg ttagactttc caaccattat 9600
gaaaacacta gcctttttcc atactattct gatcaatggt tacaggagga agcatcatgg 9660
agtatggcca cccttgaact taccgggtaa tgcttcaaag ggtctcatag aacttatgaa 9720
tgacaatact gagataagct atgaattcac acttaagcat tggaaggaag tctctcttat 9780
aaaattcaag aaatgttttg atgcagacgc aggtgaggaa ctcagtatat ttatgaaaga 9840
taaagcaatt agtgcaccaa aacaagactg gatgagtgtg tttagaagaa gcctaatcaa 9900
acagcgccat cagcatcatc aggtccccct acctaattca ttcaatcgac ggctattgct 9960
aaactttctt ggcgatgaca aattcgaccc gaatgtggag ctacagtatg taacatcagg 10020
tgagtatcta catgatgaca cgttttgtgc atcatattca ctgaaagaga aggaaattaa 10080
acctgatggt cgaatttttg caaagttgac taaaagaatg agatcatgtc aagttatagc 10140
agaatctctt ttagcgaacc atgctgggaa gttaatgaaa gagaatggtg ttgtaatgaa 10200
tcagctatca ttaacaaaat cactattaac aatgagtcag attggaataa tatccgagaa 10260
agctagaaaa tcgactcggg ataacataaa tcaacctggt tttcagaata tccagagaaa 10320
taaatcacgt cactccaagc aagtcaacca gcgagatcca agtgatgact ttgaattggc 10380
agcatctttt ttaactactg atctcaaaaa atattgttta caatggaggt accagacaat 10440
tatcccattt gctcaatcat taaacagaat gtatggttat cctcatctct ttgagtggat 10500
tcacttacgg ctaatgcgta gtacacttta cgtgggggat cccttcaacc caccagcgga 10560
taccagtcaa tttgatctag ataaagtgat taatggagat atctttattg tatcacccag 10620
aggtggaatt gaagggctat gtcaaaaggc ttggacaatg atatctatct ctgtgataat 10680
tctatctgcc acagagtctg gcacacgagt aatgagtatg gtgcagggag ataatcaagc 10740
aattgctgtc accacacgag taccaaggag cttgccgact cttgagaaaa agactattgc 10800
ttttagatct tgtaatctat tctttgagag gttaaaatgt aataattttg gattaggtca 10860
ccatttgaaa gaacaagaga ctatcattag ttcccacttc tttgtttata gcaagagaat 10920
attctatcag gggaggattc taacacaagc cttaaaaaat gctagtaagc tctgcttgac 10980
agctgatgtc ctaggagaat gtacccaatc atcatgttct aatcttgcaa ctactgtcat 11040
gagattaact gagaatggtg ttgaaaaaga tatctgtttc tatttgaata tttatatgac 11100
catcaaacag ctctcctatg atatcatctt ccctcaagtg tcaattcctg gagatcagat 11160
cacattagaa tacataaata atccacatct ggtatcacga ttggctcttc tgccatctca 11220
gctaggaggt ctaaactacc tgtcatgtag taggctgttc aatcgaaaca taggcgaccc 11280
ggtggtttcc gcagttgcag atcttaagag attaattaac tcaggatgta tggattactg 11340
gatcctttat aatttattag gtagaaaacc gggaaacggc tcatgggcta ctttagcagc 11400
tgacccgtac tcaataaaca tagagtatca atacccccca actacagctc ttaagaggca 11460
cacccaacaa gctctgatgg aactcagtac aaatccaatg ttacgtggca tattctctga 11520
caatgcacag gcagaagaaa ataatcttgc tagatttctc ctggataggg aggtgatctt 11580
tccacgtgta gctcacataa tcattgagca aaccagtgtc gggaggagaa aacagattca 11640
aggatatttg gattcaacta gatcgataat gaggaaatca ctagaaatta agcccttgtc 11700
caataggaag cttaatgaaa tactagatta caacatcaat tacctagctt acaatttggt 11760
attactcaag aatgctattg aacctccgac ttatttgaag gcaatgactc ttgaaacatg 11820
tagcatcgac attgcaagga gcctccggaa gctttcctgg gccccactct tgggtgggag 11880
aaatcttgaa ggattagaga caccagatcc cattgaaatt actgcaggag cattaattgt 11940
tggatcaggc tactgtgaac agtgtgctgc aggagacaat cgattcacat ggtttttctt 12000
gccatctggt atcgagatag gaggggatcc ccgtgataat cctcctatcc gtgtaccgta 12060
cattggctcc aggactgatg agaggagggt agcctcaatg gcatacatca ggggtgcctc 12120
gagtagccta aaagcagctc ttagactggc aggagtgtac atctgggcat tcggagatac 12180
tctggaaaat tggatagatg cactggattt gtctcatact agagttaaca tcacacttga 12240
acagctgcaa tccctcaccc cacttccaac ctctgccaat ctaacccatc ggttggatga 12300
tggcacaact accctaaagt ttactcctgc gagctcttat accttttcaa gtttcactca 12360
tatatcaaat gatgagcaat acctgacaat taatgacaaa actgcagatt caaatataat 12420
ctaccaacag ttaatgatca ctggactcgg aatcttagaa acatggaata atcctccaat 12480
caatagaaca ttcgaagaat ctaccctaca tttgcacact ggtgcatcat gttgtgtccg 12540
acctgtggac tcctgcatca tctcagaagc attaacagtc aagccacata ttacagtacc 12600
gtacagcaat aaatttgtat ttgatgaaga cccgctatct gaatatgaga ctgcaaaact 12660
ggaatcgtta tcattccaag cccagttagg caacattgat gctgtagata tgacaggtaa 12720
attaacatta ttgtcccaat tcactgcaag gcagattatt aatgcaatca ctggactcga 12780
tgagtctatc tctcttacta atgatgccat tgttgcatca gactatgtct ccaattggat 12840
cagtgaatgc atgtatacca aattagatga attatttatg tattgtgggt gggaactact 12900
attagaacta tcctatcaaa tgtattatct gagggtagtt gggtggagta acatagtgga 12960
ttattcttac atgatcctaa gaaggatacc tggtgcagcg ttaaacaatc tggcatctac 13020
attaagtcat ccaaaacttt tccgacgggc tatcaactta gatatagttg cccccttaaa 13080
tgctcctcat tttgcatctc tggactacat caagatgagt gtggatgcaa tactctgggg 13140
ctgtaaaaga gtcatcaatg tgctctccaa tggaggggac ttagaattag ttgtgacatc 13200
tgaagatagt cttattctca gtgaccgatc catgaatctc attgcaagga aattaacttt 13260
attatcactg attcatcata atggtttgga attaccaaag attaaggggt tctcacctga 13320
tgagaagtgt ttcgctttga cagaattttt gaggaaagtg gtgaactcag ggttgagttc 13380
aatagagaac ctatcaagtt ttatgtacaa tgtagagaac ccacggcttg cagcattcgc 13440
cagcaataat tactacctga ccagaaaatt atcgaattca atacgagata ctgagtcggg 13500
tcaagtagcg gtcacctcat attatgaatc attagaatat attgatagtc ttaagctaac 13560
cccacatgtg cctggtacct catgcattga ggatgatagt ctatgtacaa atgattacat 13620
aatctggatc atagagtcca atgcaaactt ggagaagtat ccaattccaa atagccctga 13680
ggatgattcc aatttccata actttaagtt gaatgctcca tcgcaccata ccttacgccc 13740
attagggtta tcatcaactg cttggtataa gggtataagc tgttgcaggt accttgagcg 13800
attaaagcta ccacagggtg atcatttata tattgcagaa ggtagtggtg ccagtatgac 13860
aatcatagaa tacttattcc caggaagaaa gatatattac aattctttat ttagtagtgg 13920
tgacaatccc ccacaaagaa attatgcacc aatgcctact cagttcattg agagtgtccc 13980
atacaagctc tggcaagcac acacagatca atatcccgag atttttgagg atttcatccc 14040
tctatggaac ggaaatgccg ccatgactga cataggaatg acagcttgtg tagaatttat 14100
catcaataga gtcggcccaa ggacttgcag tttagtacat gtagatttgg agtcaagtgc 14160
aagcttaaat caacaatgcc tgtcaaagcc gataattaat gctattatca ctgctacaac 14220
tgttttgtgc cctcatgggg tgcttattct gaaatatagt tggttgccat ttactagatt 14280
tagtactttg atcactttct tatggtgcta ctttgagaga atcactgttc ttaggagcac 14340
atattctgat ccagctaatc atgaggttta tttaatttgt atccttgccg acaactttgc 14400
attccagact gtctcgcagg caacaggaac ggcgatgact ttaaccgatc aagggtttac 14460
tttgatatca cctgaaagaa taaatcagta ttgggatggt cacttgaagc aagaacgtat 14520
cgtagcggaa gcaattgata aggtggttct aggagaaaat tctctattca attcgagtga 14580
taatgaatta atcctcaaat gtggagggac accaaatgca cggaatctta tcgatatcga 14640
gccagtcgca actttcatag aatttgaaca actgatctgc acaatgttga caacccactt 14700
gaaggaaata attgatataa caaggtctgg aacccaggat tatgaaagtt tattactcac 14760
tccttacaat ttaggtcttc ttggtaaaat cagtacgata gtgagattat taacagaaag 14820
gattctaaat catactatca ggaattggtt gatcctccca ccttcgctcc ggatgatcgt 14880
gaagcagaac ttggaattcg gcatattcag gattacttcc atcctcaact ctgatcgatt 14940
cctgaagctt tctccaaata ggaaatactt gattacacaa ttaactacag gctacattag 15000
aaaattgatt gagggggatt gtaatatcga actaactaga cctatccaaa agcaaatctg 15060
gaaagcatta ggttgtgtag tctattgtca cgatccagta gatcaaaggg aatcaacaga 15120
gtttattgat ataaatatta atgaagaaat agaccgcggg atcgatggcg aggaaatcta 15180
aatatatcaa gaatcagaat tagtttaaga aaaaataaga ggattaatct tggttttccc 15240
cttggt 15246

Claims (14)

1. A canine parainfluenza virus strain, wherein the full-length cDNA of the genome of the canine parainfluenza virus strain is a sequence shown in SEQ ID No.1 or a sequence obtained by replacing CDS in the SEQ ID No.1 with a degenerate sequence thereof.
2. The canine parainfluenza virus strain of claim 1, wherein the canine parainfluenza virus strain is CPIV HeN0718 strain, and the genome full-length cDNA of the canine parainfluenza virus strain CPIV HeN0718 strain is a sequence shown in SEQ ID No. 1.
3. A vaccine composition comprising an immunizing amount of the canine parainfluenza virus strain of claim 1 or a culture-inactivated antigen thereof and a veterinarily acceptable carrier.
4. The vaccine composition according to claim 3, wherein the canine parainfluenza virus strain antigen is an inactivated antigen of the canine parainfluenza virus strain CPIV HeN0718 strain or a culture thereof, and the inactivated antigen content of the canine parainfluenza virus strain CPIV HeN0718 strain or the culture thereof is 10 before inactivation4.0-107.0TCID50/ml。
5. The vaccine composition according to claim 3, wherein the inactivated antigen content of the canine parainfluenza virus strain CPIV HeN0718 strain or the culture thereof is 10 before inactivation5.0-106.0TCID50/ml。
6. The vaccine composition according to claim 3, wherein the inactivated antigen content of the canine parainfluenza virus strain CPIV HeN0718 strain or the culture thereof is 10 before inactivation6.0TCID50/ml。
7. The vaccine composition of claim 3, wherein said culture of canine parainfluenza virus strain is a culture cultured for 1-18 passages.
8. The vaccine composition of claim 3, wherein said culture of canine parainfluenza virus strain is a culture cultured for 6-18 passages.
9. The vaccine composition of claim 3, wherein the veterinarily acceptable carrier is an adjuvant, which is GelA adjuvant;
the concentration of the adjuvant ranges from 5% to 50% V/V.
10. The vaccine composition of claim 9, wherein the adjuvant is at a concentration of 5% V/V.
11. The vaccine composition of claim 3, wherein the vaccine composition further comprises a canine distemper virus antigen, and/or a canine parvovirus antigen.
12. The vaccine composition of claim 11, wherein the vaccine composition comprises a pre-inactivation 10 content4.0-107.0TCID50The inactivated antigen of the/ml canine parainfluenza virus strain CPIV HeN0718 strain with the content of 10 before inactivation4.0-106.0TCID50The inactivated antigen of CDV C/HN/001 strain of canine distemper virus of/ml and/or the content of the inactivated antigen is 10 before inactivation6.0-108.0TCID50The inactivated antigen of the canine parvovirus CPV S0425 strain in/ml and the adjuvant of 5% V/VGEL A.
13. The vaccine composition of claim 12, wherein the vaccine composition comprises a pre-inactivation 10 content6.0TCID50The inactivated antigen of the/ml canine parainfluenza virus strain CPIV HeN0718 strain with the content of 10 before inactivation5.0TCID50The inactivated antigen of CDV C/HN/001 strain of canine distemper virus of/ml and/or the content of the inactivated antigen is 10 before inactivation7.0TCID50The inactivated antigen of the canine parvovirus CPV S0425 strain in/ml and the adjuvant of 5% V/VGEL A.
14. Use of a vaccine composition according to any one of claims 3 to 13 in the manufacture of a medicament for the prevention and treatment of a disease associated with canine parainfluenza virus infection.
CN201610946235.9A 2016-11-02 2016-11-02 Canine parainfluenza virus strain and application thereof Active CN108018261B (en)

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