CN114958780B - Bovine Aichivirus D virus isolate and application thereof - Google Patents
Bovine Aichivirus D virus isolate and application thereof Download PDFInfo
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- CN114958780B CN114958780B CN202210630701.8A CN202210630701A CN114958780B CN 114958780 B CN114958780 B CN 114958780B CN 202210630701 A CN202210630701 A CN 202210630701A CN 114958780 B CN114958780 B CN 114958780B
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Abstract
The invention discloses a bovine Aichivirus D virus isolate and application thereof, belonging to the technical fields of biotechnology and virology, wherein the classification of the virus isolate is named as Yak Aichivirus D virus BKV/Yak/DF77/2021/CHN, and the preservation number is as follows: CCTCC NO: V202228. The strain provided by the invention has higher titer, can generate higher antibodies after being prepared into an inactivated vaccine, can be used as a candidate strain for vaccine development, and has very good application prospect in the aspects of prevention and treatment and diagnosis of diseases caused by bovine Aichivirus D virus.
Description
Technical Field
The invention relates to the fields of biotechnology and virology, in particular to a bovine Aichivirus D virus isolate and application thereof.
Background
Kubuvirus (kobuvirus) belongs to the Picornaviridae family (Picornaviridae), and the viral genome consists of a 5'utr, a large Open Reading Frame (ORF) and a 3' utr region. Kobuvirus virus is a causative agent of diarrhea, which has strong genetic diversity, high recombination frequency and RNA replication substitution rate. Currently, 6 kobuviruses (Aichiviruses A-F) and 3 unclassified kobuviruses (Gray, norway and bats) are known. Aichiviruses are a novel genus of the family Picornaviridae, and the presence of this virus, found in humans and in a variety of animals and their environment, is a novel causative agent of diarrhea in animals. At present, the virus has been detected in more than ten countries in the world including China, and has wide regional distribution.
Among the Aichiviruses A-F, aichivirus A is found in cats, sheep, and mice, aichivirus B is found in cattle, ferrets, and sheep, and Aichivirus C is found in pigs only. While known viruses for cattle infection currently include Aichivirus B (AiV-B) and Aichivirus D (AiV-D), aiV-B has been popular in cattle breeding clusters in China, and another virus capable of infecting cattle AiV-D has not been reported in China, keha-moAbi et al have been found in Tibetan sheep diarrhea manure by using a virus metagenomic method in 2021, and successfully obtained complete Kobuvirus genome from diarrhea manure by using RT-PCR. The genome sequence length is 8485 nucleotides, the genome structure of the small RNA virus accords with the standard, the homology of the separated genome with the Aichivirus D1 genotype 1-22-KoV strain and the Aichivirus D2 genotype 2-44-KoV strain is 62.9% and 77.8%, respectively, and the newly discovered kutivirus belongs to Aichivirus D (Keha-mo Abi, et al identification of a novel Aichivirus D in slide. Effect, genetics and Evolution 91 (2021) 104810 according to the species classification standard of the International Commission for viral classification (ICTV). There is no report of successful isolation of Aichivirus D from cattle.
Therefore, the epidemic strain Aichivirus D in China is urgently needed to be separated from cattle so as to develop a vaccine to prevent the disease, and the economic loss of the virus to the cattle industry is reduced.
Disclosure of Invention
The invention aims to provide an isolated strain of bovine Aichivirus D virus and application thereof, wherein the isolated strain of the Aichivirus D virus (namely BKV/Yak/DF77/2021/CHN strain) can generate higher-level antibodies after being immunized by a vaccine, can be widely used for differential diagnosis of clinical bovine Aichivirus D virus vaccine and related diseases, and fills the blank that no technology for preparing products for preventing or treating the diseases caused by the bovine Aichivirus D virus is independently developed in China at present.
In order to achieve the purpose of the invention, the technical scheme adopted is as follows:
in a first aspect, the invention provides an bovine Aichivirus D virus isolate, which is characterized in that the isolate has a microorganism preservation number of CCTCC NO. V202228.
In a second aspect, the present invention provides the use of a bovine Aichivirus D virus isolate as described above in the preparation of a product for the prophylaxis and/or treatment of a disease caused by bovine Aichivirus D virus.
The term "prevention" according to the invention refers to all actions of inhibiting diarrhea or delaying onset of a disease in a cow by administering a vaccine composition according to the invention.
The term "treatment" refers to all actions that lead to a alleviation or improvement of symptoms caused by a bovine diarrhea virus infection by administration of a vaccine composition according to the invention.
Further, the product is a vaccine or a medicament.
Further, the vaccine is selected from one or more of inactivated vaccine, attenuated live vaccine, genetic engineering vaccine and polypeptide vaccine.
The term "vaccine" is defined broadly herein to refer to any type of biological substance in an administrable form that is capable of stimulating an immune response in an animal vaccinated with the vaccine. Vaccines in general may be based on the virus itself (e.g. inactivated/inactivated or attenuated). In one embodiment of the invention, the vaccine (immunogenic composition) preferably comprises inactivated/inactivated form of viral material, or an antigenic portion of a virus present as a subunit vaccine.
In a third aspect, the present invention provides an anti-bovine Aichivirus D virus antibody prepared from the aforementioned bovine Aichivirus D virus isolate.
In a fourth aspect, the present invention provides a medicament comprising an anti-bovine Aichivirus D virus antibody as described above.
In a fifth aspect, the present invention provides the use of the bovine Aichivirus D virus isolate or the anti-bovine Aichivirus D virus antibody described above in the preparation of a reagent for detecting a disease caused by bovine Aichivirus D virus.
In a sixth aspect, the invention provides a vaccine composition comprising an effective amount of the aforementioned bovine Aichivirus D virus isolate.
The term "effective amount" as used herein in the context of a composition means an amount of an immunogenic composition that is capable of inducing an immune response that reduces the incidence of infection or reduces the severity of infection or reduces the occurrence of disease in an animal. Specifically, an effective amount refers to Colony Forming Units (CFU) per dose. Alternatively, in the context of treatment, the term "effective amount" refers to an amount of treatment sufficient to achieve the following objectives: reducing or alleviating the severity or duration of a disease or disorder (or one or more symptoms thereof), preventing the progression of a disease or disorder, causing regression of a disease or disorder, preventing the recurrence, development, onset, or progression of one or more symptoms associated with a disease or disorder, or enhancing or improving the prophylactic or therapeutic effect of another therapeutic or therapeutic agent. The exact amounts of ingredients used and the compositions to be administered will vary depending upon factors such as the type of disease being treated, the type and age of the animal to be treated, the manner of administration, and other ingredients in the composition.
Further, the vaccine composition further comprises a pharmaceutically or veterinarily acceptable carrier.
Pharmaceutically or veterinarily acceptable carriers "include any or all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, bacteria and antifungal agents, isotonic agents, adsorption delaying agents, and the like. In some preferred embodiments, and in particular those comprising a lyophilized immunogenic composition, the stabilizers for use in the present invention comprise stabilizers for lyophilization or freeze drying.
"diluents" may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. Stabilizers include the basic salts of albumin and ethylenediamine tetraacetic acid, and the like.
In some embodiments of the invention, the vaccine composition of the invention further comprises an adjuvant. The adjuvants include oil adjuvants including, but not limited to, animal, vegetable or mineral oils, white oils, squalane or squalene, and drekey oil (Drakeoil). The above oil adjuvant may be either derived or obtained synthetically.
In the present invention, the vaccine composition includes, but is not limited to, an oil-in-water emulsion, a water-in-oil emulsion, or a double emulsion; the double emulsion is represented by an oil-in-water-in-oil emulsion.
In a seventh aspect, the invention provides a method for preparing the vaccine composition, culturing and proliferating bovine Aichivirus D virus isolate, and inactivating to prepare a vaccine product.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs at the time of its filing. The meaning and scope of the terms should be clear; however, if there are any potentially ambiguous situations, the definitions provided herein take precedence over any dictionary or external definitions. In this document, the use of "or" means "and/or" unless stated otherwise. In addition, the use of the term "including," as well as other forms such as "comprising" and "containing," is not limiting.
The beneficial effects are that:
(1) The invention detects the yak Aichivirus D virus in the diarrhea yak fecal sample and obtains a complete genome, and the invention confirms the existence of the yak Aichivirus D virus in China for the first time, so that the prevention and control of the yak Aichivirus D virus have more pertinence and effectiveness, and the invention has important value in the application research of inactivated vaccine and attenuated vaccine of the yak Aichivirus D virus.
(2) The invention uses the isolated strain of the yak Aichivirus D virus to prepare the vaccine for preventing diarrhea caused by the yak Aichivirus D virus, and the vaccine has good safety and immunogenicity after animals are immunized by the vaccine, has no adverse reaction in observation time, and can stimulate organisms to generate high-titer specific serum neutralizing antibodies.
Drawings
FIG. 1 is a schematic diagram of a BKV/Yak/DF77/2021/CHN strain-induced Vero cytopathy; wherein A: normal Vero cells; b: plaque purification yields diseased Vero cells; c: indirect immunofluorescence identification; d: transmission electron microscope
FIG. 2 is a PCR assay of an isolate of the invention; wherein, gel electrophoresis application of sample order does in proper order: lane 1: a positive control; lane 2: a negative control; lane 3: BKV/Yak/DF77/2021/CHN; m: marker II is 1200bp, 900bp, 700bp, 500bp, 300bp and 100bp from top to bottom in sequence;
FIG. 3 is a BKV/Yak/DF77/2021/CHN specificity and purity test of the present invention; wherein, lane 1: aiV D positive control; lane 2: BKV/Yak/DF77/2021/CHN AiV D detection; lane 3: aiV D negative control; lane 4: BRV positive control; lane 5: BRV negative control; lane 6: BKV/Yak/DF77/2021/CHN BRV detection; lane 7: a BcoV positive control; lane 8: a BcoV negative control; lane 9: BKV/Yak/DF77/2021/CHN BcoV detection; lane 10: BEV positive control; lane 11: BEV negative control; lane 12: BKV/Yak/DF77/2021/CHN BEV detection; m: mark II is 1200bp, 900bp, 700bp, 500bp, 300bp and 100bp from top to bottom in sequence;
FIG. 4 is a graph showing the measurement of neutralizing antibody levels after immunization of calves with an inactivated vaccine prepared from the isolated strain of the present invention.
Detailed Description
The following will describe embodiments of the present invention in detail by referring to examples, so that the implementation process of how to apply the technical means to solve the technical problems and achieve the technical effects of the present invention can be fully understood and implemented.
The method used in the present invention may be a method commonly used in the field of virus research, and is not limited to the specific description of the embodiments of the present invention. The experimental procedures used in the examples below were, unless otherwise specified, either in accordance with conventional experimental conditions or in accordance with the manufacturer's instructions. The reagents, materials, etc. used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 isolation and identification of yak AiV D Virus
1.1 isolation of yak AiV D Virus
Firstly, observing the disease condition of a cattle group, and collecting a cow dung sample which is preliminarily diagnosed as diarrhea. Fecal samples were treated with PBS (100U/mL penicillin, 100g/mL streptomycin) 1: and 5, diluting. Centrifuging at 3000r/min for 10min to obtain supernatant, and filtering and sterilizing with 0.22 μm filter membrane to obtain the disease material treatment liquid.
Inoculating the above disease material treatment solution into vigorous monolayer Vero cells on 6-well culture plate, inoculating 0.5mL of each well, and simultaneously setting negative control well containing 5% CO at 37deg.C 2 The culture was performed in an incubator for 1 hour, the culture solution was discarded, 2mL of a maintenance solution (DMEM containing 1% fetal bovine serum) was added, and the mixture was subjected to a reaction at 37℃with 5% CO 2 Culturing in an incubator, observing cytopathy every 12h, sterilizing after 96h, repeatedly freezing and thawing the cytotoxin for 3 times, and inoculating Vero cells again, and carrying out passage until typical cytopathy appears.
After the Vero monolayer cells are inoculated with the disease treatment fluid, cytopathy does not appear in the first two generations, viruses are transmitted to the 4 th generation in a blind way, local cytopathy begins to appear in diarrhea fecal samples BKV/Yak/DF77/2021/CHN, cell cultures are harvested, typical cytopathy phenomena are generated after the Vero cells are inoculated for 48 hours again, the typical cytopathy phenomena are mainly represented by particle deformation of infected cells, cell aggregation and stacking, cytopenia and the like (see figure 1), most of cells fall off after the cytopathy is completely treated, other cytopathy does not appear, and cells of a control group grow well.
1.2 RT-PCR identification of yak AiV D virus
1.2.1 viral PCR detection
The AiV D detection primers in Table 1 were synthesized and sent to Shanghai Biotechnology Co., ltd, and the primer sequences were as follows:
TABLE 1 AiV D detection primers
Taking 400 mu L of Vero cell culture of the isolated strain as an extraction sample of viral RNA, extracting RNA of feces by referring to Trizol kit (RNAisoPlus) instruction of Shanghai Biotechnology, inc., taking 400 mu L of supernatant, adding 700 mu L of Trizol, and standing at room temperature for 10min; 200 mu L of chloroform is added, and the mixture is vigorously shaken for 15s, and the supernatant is taken; adding isopropyl alcohol with the same volume, fully reversing and uniformly mixing, and standing at room temperature for 10min; centrifuging at 12000r/min at 4deg.C for 15min, discarding supernatant, and adding 1mL 750mL/L (prepared with sterilized water treated with DEPC) ethanol; centrifuging at 4deg.C for 5min at 12000r/min, discarding supernatant, dissolving the precipitate with 15 μL of DEPC-treated sterile water, and placing in a refrigerator at-80deg.C for use.
The RNA was reverse transcribed into cDNA using the Prime Script TMRT kit. The reaction system of RT-PCR amplification takes cDNA as a template as follows: 2. Mu.L of template cDNA, quick Taq HS DyeMix. Mu.L, 1.0. Mu.L of each of the upstream primer and the downstream primer, and the remainder of the primer was composed of ddH 2 O is added up to 20 mu L.
The reaction procedure was as follows: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30s, annealing at 52℃for 30s, elongation at 68℃for 1min for 35 cycles; extending at 72℃for 8min.
The amplified product is detected by agarose gel electrophoresis, the result shows that the isolate obtains a specific band with the size of about 568bp (see figure 2), the PCR product is sent to Shanghai Biotechnology Co Ltd for sequencing analysis, the nucleotide sequence is shown as SEQ ID NO.1, the determination result and the published AiV D sequence of NCBI are subjected to genotyping analysis, and the result shows that the isolate BKV/Yak/DF77/2021/CHN belongs to the AiV D2 subtype.
1.2.2 purity detection
400. Mu.L of Vero cell virus liquid was taken as an RNA extraction sample, fecal RNA was extracted by referring to Trizol kit (RNAisoPlus) instructions from Shanghai Biotechnology, inc., and RNA was reverse transcribed into cDNA using Prime Script TMRT kit. Exogenous virus detection is carried out on Bovine Rotavirus (BRV), bovine coronavirus (BsoV) and Bovine Enterovirus (BEV) by taking the synthesized cDNA as a template and applying an RT-PCR detection method.
TABLE 2 exogenous viral detection primer sequences
The reaction system of RT-PCR amplification is as follows: 2. Mu.L of template cDNA, quick Taq HS DyeMix. Mu.L, 1. Mu.L of each of the upstream primer and the downstream primer, and the remainder of the primer was composed of ddH 2 O is added up to 20 mu L. The reaction procedure was as follows: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30s, annealing at 52℃for 30s, elongation at 68℃for 1min for 35 cycles; extending at 72℃for 8min. The AiV D isolate was negative by RT-PCR detection and by exogenous virus detection BEV, bcoV, BRV.
1.3 Virus plaque purification
Serial 10-fold dilution of virus liquid to 10 -6 Inoculating the virus into a single-layer Vero cell 6-well culture plate with good growth, inoculating 100 mu L of virus diluent to each well, inoculating 2 wells for each dilution, setting blank control wells at the same time, placing the blank control wells into a cell culture box for adsorption for 1h, and shaking every 20min to uniformly distribute the virus. Removing virus liquid in 6-well plate after adsorption, adding 2mL of 2% methylcellulose culture medium containing 4% FBS into each well, spreading on cell surface, solidifying at room temperature, and standing at 37deg.C with 5% CO 2 Is continuously cultured in the cell incubator. After a single plaque appears at the appropriate dilution, a sterile gun tip is used to select for typical plaque reproducibilityCloning, inoculating monolayer Vero cells, culturing and proliferating, repeating the above steps for 3 times. And 3 times of purification, and then mass propagation of virus liquid is used as seed virus.
1.4 determination of viral titres
Spreading Vero cell suspension with good growth state on 96-well plate, serial 10-fold dilution of cell virus culture solution, adding into 96-well cell plate, each dilution of 8 wells, 100 μl of each well, placing into 37 deg.C cell incubator containing 5% CO for 1 hr, discarding virus solution, adding 1% fetal bovine serum DMEM culture solution, and 37 deg.C cell incubator containing 5% CO 2 Culturing in a cell incubator, observing pathological conditions of cells in 2-3 days, and performing TCID on the isolated strain according to a Reed-Muench calculation method 50 Determination, result BKV/Yak/DF 77/2021/CHN=10 -7.57 /0.1mL。
Preservation information:
the collection number of the isolated strain of the yak Aichivirus D virus is CCTCC NO: V202228, and the classification of the virus is named as: yak Aichivirus D virus BKV/Yak/DF77/2021/CHN, latin literature name is Yak Aichivirus D, was deposited at the China center for type culture Collection at 2022, month 4 and 10: in the Wuhan university of No. 299 of Wuhan in Wuhan district of Wuhan, hubei province (first additional face of Wuhan university).
EXAMPLE 2 development of AiV D inactivated vaccine
2.1 propagation of Virus
Taking vero monolayer cells with good growth state, washing with Hanks for 2 times, inoculating the vaccine candidate strain DF77 cell culture solution with good immunogenicity to vero cells, adding DMEM cell maintenance solution containing 1% fetal bovine serum, and placing at 37deg.C and containing 5% CO 2 When the cytopathy is about 80%, harvesting the virus culture solution, repeatedly freezing and thawing for 3 times, and centrifuging at 4 ℃ at high speed to obtain virus supernatant serving as the virus for the vaccine.
2.2 preparation of inactivated Virus vaccine
The vaccine candidate strain DF77 cell culture supernatant with good immunogenicity is fully and uniformly mixed with the AiV D virus liquid by using beta-propiolactone with the final concentration of 0.5 percent, and the mixture is inactivated for 6 hours in a shaking table at 37 ℃. Taking two inactivated virus solutions, respectively inoculating the virus solutions to vero cells for inactivation and detection, carrying out blind transmission on the vero cells for two generations, culturing for 48-72 hours in each generation, observing cytopathic conditions, and simultaneously setting positive virus control and blank control.
Cells of the detection group and the negative control group inoculated with the inactivated virus liquid are free of CPE, typical AiV D cytopathy appears in the positive control group, and 2-generation blind transmission is carried out on the detection group, and CPE is not seen yet, so that the inactivation of the detected inactivated virus liquid is complete.
Finally, mixing the inactivated AiV D with the 201 adjuvant in equal volume, stirring the mixture and fully and uniformly mixing the mixture, emulsifying the mixture in an emulsifying machine for 5 minutes to prepare the inactivated vaccine of water-in-oil-in-water, subpackaging the inactivated vaccine in a transparent glass bottle, observing that the vaccine is milky uniform suspension, sucking a drop of the inactivated vaccine into a beaker containing water by using a syringe, uniformly dispersing the vaccine in the whole beaker, and centrifuging the inactivated vaccine for 5 minutes at 3000r/min, wherein layering phenomenon is not seen, and the vaccine is qualified in inspection and can be used for subsequent experiments.
TABLE 3 Table 3
Selecting 6 healthy calves with 3-6 month-old AiV D antigen and antibody negative. The animals are randomly divided into 5 groups of experiment groups, 1 group of control group, and whether clinical abnormality exists or not is observed every day, and all animals are sampled for detecting the level of virus antibody. First immunization by neck muscle vaccination with inactivated vaccine (DF 77); the blank group was inoculated with the same dose of virus-free cell fluid. The second immunization was performed 21 days after vaccination in the same manner and dosages as described above. The safety test result shows that the DF77 virus strain is safe to calves and has no side effect. Blood was collected at various times after vaccination, and the level of neutralizing antibodies after calf immunization was determined using an antibody neutralization test (fig. 4). The result of the immune test shows that the DF77 strain can induce high-level antibody after 7 days of immunization, and the SNT titer reaches 1400U/ml, thus being used as a candidate strain for vaccine development.
While the foregoing description illustrates and describes several preferred embodiments of the invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the invention described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Sequence listing
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cttctgctcc ctctaccacc tctgctgctt ttgctgatcc ccgtgggact gcgctggagt 3780
actacaagct ccatgcgaag gctcaccccc aacttcccat tgccctcgcc cgtcgccagt 3840
gccaagacac tgaggtgccg tctctgctca acccagactc gcgcatctac attgtcaagg 3900
cgcagcgccc cacctatgtt cattgggcta tccgtgcggt ccatcccgat ggatcctcgg 3960
aacaaatttc cctctcccgt tccggcattg gctgcgtgat cgcctacgag gaatgtgaag 4020
gtgaactcta ccaggaggtc gctcctggtt gctgggtcac cgccacttgc ctcgttggcg 4080
agccgtggga atacaatgct gagaacaact gcaccacctt tgtctccaat gtgactggtg 4140
tctctctccc caacaccggt tactccctca tctttggggt tttcgctctc tctgccgtca 4200
gcctggtggc cgcgcaagcg ctctcaggcc gccacgccca acgtcaggga gactctgatt 4260
cttccgcttc ctttgcatct gctcgcccca tccccgctcc ccgttctctc acttgcacca 4320
tcttctctac ttctgagtct gacgccccta ggccgcgccc gcgtcgccaa accaggcgat 4380
gccgccggca aagtacgtcg gattctgact ctgacccctc ttggactgca actccccgtc 4440
tcatcaagga ggcgacgtct gccgcccgct acgtggcgga cacggcgaag gagctcgagc 4500
tgaaggccac atccaacaac ctgatgctct gctcctccga cctccgctgc gcggcggaca 4560
aagtttccgc ttccgttgat gggttccggg agtttctcgg ctcttggtcc tcctccttca 4620
aggataatgt cggtgatgcc gtctccagcg gtattgccac cttcctgaag tgggtcgcca 4680
agtgttttgg ctatctctta gtcatttttg gttcccctac ccccatgtct ctcactggtg 4740
ttattcttct cctctgtgct gatctctctc ccgacattgc tgggttcttc accaaagttc 4800
ccaacgctgt tggcgctctt tacttttgga ttgccaacaa attaggcctc tcagtctcag 4860
cccaggagtg cgctgaacag ggtgtccaac atcagagcgg tcttggcgac ttcaacacct 4920
tcgccaacgc cgtgaagaac atggaatacc ttgccagtaa atcctgggaa tggactgcca 4980
aattacttga ttggattcag ggcaaagtca aaactgaccc aaacctccaa ttggccaatg 5040
ctcacgacga gatcctccga ttttatcgtg agtccattga agcgctatcc gcagttccca 5100
acgctgcggc ggccgctgat tcaatttcca ggctgcaaga attggcaaaa attgcagccg 5160
atgccaaatc tggccctcac tcaacataca ttcagcaatc catcaaaaat ttcaccaccg 5220
tcctcaccaa ggctgggaag cgctactcag gtaaccgccc cgaacccttc gtggtgtacc 5280
tgactggcgc ccctggctgc ggtaaatccc tccttgcttc catccttgcc gcaacactcg 5340
cgaaacgcct cggtggtagc gtcgacgacg tctacgcccc cgccgccgcc gactgcgagt 5400
tctatgacgg gtatactggc cagtccgtcc acttcatcga cgacattggc caggaccctg 5460
aaggcaagga ctggaagaat ttccccaatc tcgtctgctc tgctcccttc atcctcccca 5520
tggccgcgat cgaagacaag ggcacttatt acacatccag ggtaataatc gccacctcaa 5580
actttgatcg cgcgaacgac cgcgctgccc gttgcatgcc cgccctggaa cgcagactcc 5640
acctgcgtat acagatcggt gcacggggtg accggaagtt caacgccgaa gatgccctca 5700
gagacacctc cctccctaac caatctaaat acctcaccca ccactgtaaa ttctctgatc 5760
tttcctgcta caccatgacc gtcgagcgca actccatcta cagacctgac tctggcaggt 5820
ttgacacgtt cgacgagctc gtggtcattt ttcgccgcgt tgagaacaac gctggcacct 5880
ctgaaatggt gaggaacatc gtgagacaag gagatagggt gattactcga aaccccgagc 5940
acgcacctta tcacaccgtc atccacgact tgctcaccag ctctcatgtc cccgtgcctg 6000
tcgaccttgg ccgcgccata gagacaaacc agcctctatc ggtgtgtgac aagatctggc 6060
agtaccggaa acccatcttt gccaccacta ctttcatttc tgtctgttct ttccttgcaa 6120
ctctcctcta ccttgcctac cgcctgtgga aatctcgcca cgaagaggag ggcaaagagc 6180
aggctgctta ttccggtcta ccacacctct accggaaggg caagcaggct cgcccgactc 6240
ccaagccccg ctccacccag gggcccgtgg tgaggcaggg cggcgtcaca cccgccatgc 6300
ctaacatcca caggaatgtt cttcctgtcg ctgggatatg ggagaaggag tccaaccact 6360
cctccgcctt tgtccttttc tcccgctatg ttcttatccc cactcacatc gttgctggag 6420
ctccttacat caagctcggc aacgatctct ttgacactgc tactctccct gagcctcttg 6480
atcttggtcc tgaacttcaa ctctggttct tccccactct ccgccagtac aaggacatgc 6540
gccgcttcat tgggatgcat cctcacaaga ctggctgcct tctttctgca cacgggaaca 6600
actacacgta cgtccggttc tcgaattgcc ggcgtggtcc tctcgccatt ggcgggcagc 6660
acgtcatgga cggtgcctac gtttacaacg cggcaacgtt cggtggcctc tgcggctctc 6720
ccttggtcac ggatgacccc gccgggactt cgattctcgg aattcacgtg gcaggcattc 6780
ccggatgcac tggcttttct gtccctctcc atcctttcac ggagcaaatc gccgactatg 6840
caacccagca tcagtctctg atcttctcca acggctctct caaagacgag gggctccccg 6900
gtgtcaacat caacaggaag acgcgcctga gaccaagccc ggcctacggt gcgttccctg 6960
tgaagaagga accggctgct cttcgtcgtt ctgacccccg ccttgaggag ggcatcgatc 7020
tggacactca actcttcgcc aaacacaaca aaggcgacca aattgatgaa tggccctgcc 7080
ttaaggaagc tattgctctc tacacttcca ctctccctga tcacttccgg atcctcacac 7140
aagaagaggc tatccatgga actccctcca tggaaggcct cgacatgaat caggccgctg 7200
gatacccctg gaacaccttg ggaaggtcac gtcgctctct ctttgacgaa ccgaccccgg 7260
gtatctatgt accaaaacct gaacttcagg cggcaattga ccgctgtctg gagaaccctg 7320
agtacatcta ctccaccttc ctgaaagatg aactcaggcc attggataaa gtgaagaagg 7380
gcctgacccg tgccgttgag tgcgctccca tccatgccgt cattgctggc cggatgctcc 7440
ttggtggtat catcgaacac taccagggac gccccggcga gtatggcagc gcggttggct 7500
gcaaccccga ctaccactgg actcccttct tctacaaatt tgccaaatat gaaaatgtgt 7560
gggatcttga ctacaaatgc tttgatgcta ctatcccttc tgttcttctc tctgcttatg 7620
ctgattgggt gcacaaggtg acgggcgatc tgcgtgcacg ccagtacgta gaatccatcc 7680
gttggtccaa acatgtgtac gggagtgagt tatatgagat gattggcggc aatccatccg 7740
gatgtgtcgg cacatcgata atgaactcct ggtgcaacaa tgttgcggtc ctctccgcct 7800
tgatgcactg ctctggctct gatttcaacc ctcgtgatta tgaaattctt tgctatggcg 7860
atgatgtgtt gtacgcgtgt gagcccaatg tccaccctcg cgacattaag gctttctacg 7920
acaaatacac cacactcatt gtcacccctg cctctaagac ttctgacttt cctgattctt 7980
ccaccatcta tgacgtcacg tttctgaagc gctggttcgt cccggatgat atcaggggga 8040
tctacatcca ccctgtcatg actccggata cgtacgagca atcagtcatg tggatgaaag 8100
atggtgattt ccagtgtatc gttgactctc tgtcttatct ggctttccac tccggaccca 8160
aaacctacaa ggcttggtgt cagaaggtgg gagatcaggc ccggctccac ggagtcgacg 8220
tccatttcct gccctatgaa ttcctccagg ctaagtgggt taacttggtt tcggcgtga 8279
<210> 2
<211> 20
<212> DNA
<213> Synthesis (2)
<400> 2
ccaccaggta tgaattggac 20
<210> 3
<211> 20
<212> DNA
<213> Synthesis (3)
<400> 3
cgccatctga gtgattactc 20
<210> 4
<211> 18
<212> DNA
<213> Synthesis (4)
<400> 4
cgagttgaac acccagat 18
<210> 5
<211> 18
<212> DNA
<213> Synthesis (5)
<400> 5
gagacgggca tctacact 18
<210> 6
<211> 19
<212> DNA
<213> Synthesis (6)
<400> 6
agcaacactg gattgtgcc 19
<210> 7
<211> 18
<212> DNA
<213> Synthesis (7)
<400> 7
ggagtagtcc gactccgc 18
Claims (10)
1. An isolated strain of bovine Aichivirus D virus, which is characterized in that the microorganism preservation number of the isolated strain is CCTCC NO. V202228.
2. Use of the bovine Aichivirus D virus isolate of claim 1 for the preparation of a product for the prevention and/or treatment of diseases caused by bovine Aichivirus D virus.
3. The use according to claim 2, wherein the product is a vaccine or a medicament.
4. The use according to claim 3, wherein the vaccine is selected from one or more of inactivated vaccine, attenuated live vaccine, genetically engineered vaccine, polypeptide vaccine.
5. An anti-bovine Aichivirus D virus antibody prepared using the bovine Aichivirus D virus isolate of claim 1.
6. A medicament comprising the anti-bovine Aichivirus D virus antibody of claim 5.
7. Use of the bovine Aichivirus D virus isolate of claim 1 or the anti-bovine Aichivirus D virus antibody of claim 5 for the preparation of a reagent for detecting a disease caused by bovine Aichivirus D virus.
8. A vaccine composition comprising an effective amount of the bovine Aichivirus D virus isolate of claim 1.
9. The vaccine composition of claim 8, further comprising a pharmaceutically or veterinarily acceptable carrier.
10. A method of preparing a vaccine composition as claimed in claim 8 or 9, wherein the bovine Aichivirus D virus isolate is grown and inactivated to prepare the vaccine product.
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| US8486420B2 (en) * | 2005-02-15 | 2013-07-16 | The University Of North Carolina At Chapel Hill | Live virus vaccines |
| WO2007094854A2 (en) * | 2005-12-14 | 2007-08-23 | The Board Of Regents Of The University Of Oklahoma | Rna virus vaccines and methods |
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| CN113474456A (en) * | 2018-11-14 | 2021-10-01 | 博德研究所 | Droplet diagnostic systems and methods based on CRISPR systems |
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