CN116478937A - Gene III avian reovirus variant and application thereof in preparing vaccine - Google Patents
Gene III avian reovirus variant and application thereof in preparing vaccine Download PDFInfo
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Abstract
The invention discloses a gene III type avian reovirus variant strain and application thereof in preparing vaccines. The invention successfully separates 1 strain of gene III type avian reovirus variant from joint tissues of examined sick chickens, which is named ARV-HLJ21-1690401 and is preserved in China general microbiological culture collection center (CGMCC) No.45563. The result of the sigma C genetic evolution analysis shows that the variant strain is positioned on the III-type branch of the ARV gene. The variant has obvious pathogenicity to chicken embryo and SPF chicken, and can cause 90% death of inoculated chicken embryo and 100% morbidity of inoculated SPF chicken of 6 weeks old. The vaccine can be prepared into inactivated vaccine, and the immune SPF chicken can induce to generate better antibody level and can provide complete immune protection for the isotypic strains. The invention provides a new technical means for preventing the avian reovirus disease.
Description
Technical Field
The invention relates to a separated gene III type avian reovirus variant strain and also relates to application of the variant strain in preparing vaccines. The invention belongs to the field of biotechnology.
Background
In recent years, the infection condition of Avian Reovirus (ARV) is increasingly severe in China, and the ARV infection can cause diseases such as chicken viral arthritis, tenosynovitis, malabsorption syndrome and the like, so that the chicken flock has immunosuppression, reduced production performance, reduced feed utilization rate and conversion rate, the weight gain of commodity chickens is seriously influenced, and huge economic loss is caused for the poultry industry in China.
ARV is a non-enveloped double-stranded RNA virus whose genome consists of 10 segments, and the σc gene in the S1 gene segment is the most variable region in the genome compared to other gene segments, responsible for adsorption and induction of virus and cells to produce specific neutralizing antibodies against the virus, and is commonly used as a genetic marker for typing ARV epidemic strains. ARV strains can be classified into 6 genotypes according to their genetic evolution analysis.
Vaccine immunization is the most effective means for preventing and controlling ARV infection, currently commercially available vaccines are prepared based on a gene I type strain (such as S1133, 1733 and 2408), but the vaccine immunization pressure and the intensive culture density are long-term, so that viruses are continuously mutated, and researches report that the vaccine sold on the market at present can not provide complete immune protection for the mutated strain. Therefore, the epidemic situation of the ARV variant strain in China is urgently needed to be known, pathogenicity of the ARV variant strain is studied, and the immune protection effect of the current commercial vaccine on the ARV variant strain is evaluated, so that a certain guiding effect on comprehensive prevention and control of the ARV is expected.
The inventor successfully separates 1 strain of gene III type ARV variant strain from joint tissues of sick chickens examined by a certain enterprise in Heilongjiang province, which is significant for comprehensively knowing the new epidemic situation of ARV, developing novel vaccine reserve research and protecting sustainable healthy development of the breeding industry.
Disclosure of Invention
The invention aims to provide a separated gene III type avian reovirus variant strain and application thereof in preparation of inactivated vaccines.
In order to achieve the above purpose, the invention adopts the following technical means:
in 2021, in a broiler enterprise (vaccinated with commercial ARV vaccine) in the black longjiang province, a plurality of commercial broilers which have produced high-level ARV antibodies showed typical ARV characteristic symptoms such as lameness, tarsal joint swelling/deformation, and the like, and an avian reovirus strain (ARV-HLJ 21-1690401) was successfully isolated by using LMH (Leghorn male chicken hepatocellular carcinoma, LMH) cells, and its biological characteristics and genetic characteristics were revealed. The sigma C gene genetic evolution analysis shows that the isolated strain is an ARV variant strain, has highest homology with reported gene III type reference strains ISR5233 and 42563-4-2005, is in the same evolution branch, and has lower homology with other genotype strains of ARV; and vaccine strain S1133σC gene nucleusNucleotide and its coding amino acid sequence homology are 58.5% and 50.8%, respectively, suggesting that there is a large antigenic variation between the isolated strain and the commercial vaccine strain. It is reported in the literature that cross-protection effects will be reduced when the amino acid difference between the virus strain and the vaccine strain reaches or exceeds 5%. The ARV-HLJ21-1690401 variant strain identified by the invention has low homology with the amino acid sequence encoded by the sigma C gene of the main protective antigen of the current commercial vaccine strain S1133, has obvious antigenicity difference and possibly escapes the protection of the current vaccine. The LMH cell subculture results show that the variant strain can be well proliferated on LMH cells to generate typical cell fusion lesions, the virus replication titer is higher, and the virus replication titer can reach 10 after infection of 72 h 7.0 TCID 50 and/mL or more. The variant has obvious pathogenicity to chick embryo and SPF chick, can cause 90% death of inoculated chick embryo, 100% morbidity of inoculated SPF chick of 6 weeks old, and has symptoms of ARV infection such as swelling of claw pad. The strain has good immunogenicity, can be used for preparing an inactivated vaccine, can induce the immune SPF chicken to generate better antibody level, and can provide complete immune protection for the isotypic strain.
On the basis of the above study, firstly, the invention provides a variant strain of the gene III type avian reovirus, named ARV-HLJ21-1690401, classified and named avian reovirus, which is preserved in China general microbiological culture Collection center (China Committee for culture Collection), and the preservation address is in North Silu No.1, 3 of the Korean region of Beijing, china academy of sciences of microorganisms, and the preservation number is: CGMCC No.45563, the preservation time is as follows: 2023, 4 and 25 days.
The invention further provides application of the gene III type avian reovirus variant strain in preparation of medicines for preventing avian reovirus diseases.
Wherein, preferably, the medicine is an avian reovirus inactivated vaccine.
The invention further provides an avian reovirus gene III type inactivated vaccine, wherein the active ingredient of the inactivated vaccine is the inactivated gene III type avian reovirus variant.
Finally, the invention also provides a method for preparing the avian reovirus gene III type inactivated vaccine, which comprises the following steps:
adding formaldehyde with a volume ratio of 1 per mill into the F6 generation virus culture solution of the gene III type avian reovirus variant, uniformly mixing, placing at 37 ℃ for 1 time after every 1 hour of rotation and mixing, and placing for 24 hours to obtain an inactivated virus solution; tween-80 with the volume ratio of 5% V/V and inactivated virus liquid is 1:20, mixing uniformly, and emulsifying with white oil according to the volume ratio of 1:1.5, wherein the specific operation of emulsification is as follows: adding white oil into an emulsifying bottle to emulsify for 30s, and slowly adding the virus mixed solution along an emulsifying head, wherein no bubbles are generated. Regulating the emulsifying device to 6 grades, emulsifying for 3 min/time, repeating for three times, and dripping the emulsified sample into water, wherein the emulsified sample is not scattered for 5-10 s.
Compared with the prior art, the invention has the beneficial effects that:
the invention successfully separates 1 strain of avian reovirus from joint tissues of sick chickens examined by certain enterprises in Heilongjiang province of China, and the strain is named as ARV-HLJ21-1690401, and has good purity. The result of sigma C genetic evolution analysis shows that the isolated virus is located on ARV gene III branch, has a relatively short genetic distance with reference strains ISR5233 and 42563-4-2005 of gene III, has amino acid sequence homology of 83.4% and 81.7% respectively, and has a relatively long genetic distance with other genotype isolated strains. The isolated strain proliferates well on LMH cells; has obvious pathogenicity to chicken embryo and SPF chicken, can cause 90 percent death of inoculated chicken embryo, and 100 percent morbidity of inoculated SPF chicken of 6 weeks of age. The strain has good immunogenicity, can be used for preparing an inactivated vaccine, can induce the immune SPF chicken to generate better antibody level, and can provide complete immune protection for the isotypic strain. The invention provides an important theoretical basis for solving the epidemic and genetic evolution characteristics of ARV variant strains in China, and provides a new technical means for comprehensively preventing and controlling the disease in poultry farms in China.
Drawings
FIG. 1 is a diagram (100X) of cytopathic observations of ARV isolate ARV-HLJ21-1690401 infected LMH cells;
FIG. 2 is a graph showing the results of ARV isolate ARV-HLJ21-1690401 sigma C gene amplification;
wherein M is DNA molecular mass standard; 1: ARV-HLJ21-1690401;2: ARV-HeB02;3: a negative control;
FIG. 3 is a diagram of the genetic evolution analysis of the nucleotide sequence of ARV isolate ARV-HLJ21-1690401 sigma C gene;
FIG. 4 is a diagram showing the detection of exogenous viruses by ARV isolate ARV-HLJ21-1690401;
wherein M is DNA molecular mass standard; ARV-HLJ21-1690401; positive control; negative control;
FIG. 5 is a graph of the in vitro replication kinetics of ARV isolates ARV-HLJ21-1690401;
FIG. 6 is a graph showing the disease state of chicken feet at different time points after the ARV isolate ARV-HLJ21-1690401 challenge;
FIG. 7 is a graph showing the detection of the levels of antibodies in immunized SPF chickens after the ARV isolate ARV-HLJ21-1690401 has been inactivated.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below in connection with the embodiments of the present invention. It is apparent that the described embodiments are only a part of the present invention, not all embodiments, and that all other embodiments obtained by persons skilled in the art without making creative efforts based on the embodiments in the present invention are within the protection scope of the present invention.
EXAMPLE 1 isolation and identification of avian reovirus variant ARV-HLJ21-1690401 of Gene III
1. Materials and methods
1.1 Cell, experimental animal and reagent
Chicken liver cancer cells (LMH) were purchased from American Type Culture Collection (ATCC) and kept by the present laboratory. SPF chick embryos and SPF chicks were purchased from national avian laboratory animal resource libraries.
RNAiso, primeSTAR GXL DNA Polymerase, etc. are available from Takara corporation; the reverse transcription kit is purchased from TOYOBO company; the plasmid miniprep kit and the nucleic acid gel recovery kit are both purchased from Axy Prep company; DMEM/F12 cell culture broth and fetal bovine serum were purchased from Sigma.
1.2 Clinical samples and treatments
In 2021, 10 months, a certain enterprise (vaccinated with commercial ARV vaccine) in the black dragon river developed diseases characterized mainly by lameness, tarsal joint swelling and the like. Taking joint tissue or joint cavity pus of a part of sick chicken or dead chicken, adding PBS for tissue grinding, repeatedly freezing and thawing for three times, centrifuging at 4 ℃ at 8000 rpm for 10 min, collecting supernatant, filtering, sterilizing, and performing subsequent experiments.
1.3 Isolation of viruses
The supernatant of 1.2, which was ground and sterilized by filtration, was inoculated onto a 6-well plate filled with a monolayer of LMH cells at a ratio of 10% V/V, and after 1 hour of incubation, the culture broth was used for cell exchange while setting a normal cell control. At 37 ℃,5% CO 2 The culture was continued for 5 days, and the growth and pathological condition of the cells were observed every day. When cytopathy reaches 80% or above, collecting cell culture, performing continuous blind transmission for 3 passages, and storing at-20deg.C.
1.4 RT-PCR identification of isolated viruses
The culture solution of F4 generation isolated virus is taken, the total RNA of the virus is extracted by using RNAiso, and the RNA is reversely transcribed into cDNA according to the instruction of a reverse Tra Ace reverse transcription kit. A pair of specific primers, GC3F (5'-ATGGCCGGACTCACTCCATTACA-3', SEQ ID No. 1) and GC3R (5'-TTAGGTGTCGATGCCCGTACGCA-3', SEQ ID No. 2), were designed based on the ARV 42563-4-2005 strain sigma C gene sequence (DQ 872801), and amplification of sigma C gene was performed using the above cDNA as a template. PCR reaction conditions: 95 ℃ for 5 min;95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 1 min,35 cycles; the expected amplification length was 981 bp at 72℃for 10 min. After PCR amplification, the sigma C gene amplified fragment was gel recovered and ligated to pMD18-T vector, and the ligation product was transformed intoE. coli Screening is carried out in DH5 alpha cells, and positive clones are selected for sequencing.
1.5 Isolation of viral sigma C Gene homology and genetic evolution analysis
Genetic evolution and homology analysis were performed on the σc gene nucleotide sequences of the isolated strains using molecular biology software, editSeq, megAlign, etc. The reference strains used and their GenBank accession numbers are shown in Table 1.
1.6 Purity detection of isolated viruses
The DNA and RNA of the virus F4 generation virus culture broth are isolated by extraction using a conventional method, and the RNA is reverse transcribed into cDNA. The isolated ARV is tested for the presence of mycoplasma and common avian viruses, including group I avian adenovirus (FAdV), serogroup I Marek's Disease Virus (MDV), infectious anemia Virus (CIAV), reticuloendotheliosis Virus (REV), avian Influenza Virus (AIV), avian Leukemia Virus (ALV) and Infectious Bursal Disease Virus (IBDV), using the detection primers for the corresponding viruses using PCR and PT-PCR techniques.
1.7 Isolated virus titer assay and in vitro replication kinetics assay
The virus F4-generation virus solution was diluted 10-fold with DMEM containing 2% FBS and 1% SP, and the dilution was sequentially 10 -3 、10 -4 、10 -5 、10 -6 、10 -7 And 10 -8 Inoculating into 96-well plates filled with monolayer LMH cells, inoculating 16 wells per dilution, and placing at 37deg.C, 5% CO 2 After culturing in a cell incubator of 7 d, observing the occurrence of pathological changes in each well, and calculating the TCID of the virus according to the Reed-Muench method 50 。
The virus is treated by 10 4.0 TCID 50 Dose inoculates 25 cm 2 LMH cells from cell culture flasks were cultured by collecting 500. Mu.L of cell culture medium at 12 h, 24h, 36 h, 48 h, 60 h and 72 h, respectively, and TCID was performed on samples at each time point 50 And (3) drawing a one-step growth curve.
1.8 Pathogenicity test for isolated viruses
Separating F4 generation virus liquid 10 4.0 TCID 50 The egg yolk sac is inoculated with 10 SPF chick embryos of 7 days old at a dosage, the control group is inoculated with equal volume of PBS in the same way, the inoculated chick embryos are placed in a 37 ℃ incubator for culture after inoculation, the dead chick embryos in 24h are removed, the death number of the chick embryos per day is recorded, and the death rate is counted.
Taking 20 SPF chickens of 6 weeks of age, and followingThe machine is divided into 2 groups of 10 animals, one of them is inoculated with 10 6.0 TCID 50 The virus was inoculated by left single-sided paw pad, right paw pad as self-control, control group inoculated with equal volume of PBS in the same manner. The morbidity and mortality of SPF chicks are observed and recorded every day, and the mortality is counted.
1.9 Cross neutralization assay for isolated viruses
F4-generation separation virus solution was diluted with 2% FBS,1% SP DMEM to 200 TCIDs 50 100. Mu.L. Positive and negative sera of different ARV genotypes were inactivated and filtered for sterilization, followed by serial 2-fold dilution in 96-well plates. The diluted virus solution was mixed with serum in equal volume and was then treated at 37℃for 1 hour, and then 100. Mu.L of the mixed solution was inoculated into a 96-well plate filled with LMH cells, followed by continuous culture for 5 days, and the condition of lesions in each well was observed and the neutralization titer was calculated.
2. Results
2.1 Virus isolation
Inoculating LMH cells to the treated tissue grinding fluid, and after blind transmission for two generations, observing typical syncytial cytopathy, and suspending the syncytial in a culture fluid after falling off; when cultured to 84 h, cytopathy can reach more than 80 percent (figure 1).
2.2 RT-PCR identification of isolated viruses
Sigma C gene amplification using the GC3F/GC3R primer designed and synthesized in 1.4 showed that a specific band (approximately 1000 bp) corresponding to the positive control with ARV-HeB02 cDNA as a template was amplified from the virus isolate, and the negative control without cDNA template, which was identical to the expected size, was initially identified as successful in virus isolation, designated ARV-HLJ21-1690401 (fig. 2), deposited at the chinese microbiological bacterial collection center under the accession number: CGMCC No.45563.
2.3 Isolation of viral ARV-HLJ21-1690401 sigma C Gene homology and genetic evolution analysis
The homology of ARV-HLJ21-1690401 strain and sigma C gene nucleotide of ARV reference strain with different genotypes and coded amino acid thereof are compared by using EditSeq and MegAlign software. The results show that ARV-HLJ21-1690401 strain has highest homology with gene III representative strain ISR5233 (2009, israel) and 42563-4-2005 (2006, U.S.), wherein the nucleotide homology with ISR5233 strain and the coding amino acid sequence thereof are 77.2% and 83.4%, respectively, and the nucleotide homology with 42563-4-2005 strain and the coding amino acid sequence thereof are 79.3% and 81.7%, respectively; the homology with the nucleotide sequences of other genotype strains of ARV is 53.6% -58.8%, and the homology with the coding amino acid sequences is 46.4% -57.8%; the homology of the isolated strain and the vaccine strain S1133σC gene nucleotide and the coding amino acid sequence thereof is 58.5 percent and 50.8 percent respectively. In conclusion, the ARV-HLJ21-1690401 isolate was initially judged to be of genotype III based on homology alignment. The results of the genetic evolution analysis of the nucleotide sequence of the sigma C gene showed that ARV-HLJ21-1690401 strain was in the same evolutionary branch as the gene type III representative strain ISR5233, 42563-4-2005, the genetic distance was closer (FIG. 3), and the genetic distance was farther from the gene type I vaccine strain S1133 (1973, U.S.), 1733 (1997, U.S.) and 2408 (1999, china), in different evolutionary branches, and the results of the genetic evolution analysis again confirmed that the isolated virus belongs to the ARV gene III variant strain.
2.4 Exogenous viral detection of isolated virus ARV-HLJ21-1690401
The detection of mycoplasma and common avian pathogens is carried out on ARV-HLJ21-1690401 virus culture solution by using a PCR or RT-PCR method, and the result shows that the detection of specific bands exists in RT-PCR amplified products of ARV, and the detection is positive, and the detection is negative when no relevant specific bands exist in other pathogenic amplified products, thus indicating that the purity of the separated virus is good (figure 4).
2.5 In vitro replication kinetics assay to isolate virus ARV-HLJ21-1690401
ARV-HLJ21-1690401 replicated well in LMH cells, TCID thereof 50 Is 10 7.53 /ml. Based on TCID of samples at different time points after LMH infection by virus 50 As a result of the measurement, the replication kinetics of the strain was plotted (FIG. 5). As can be seen from the graph, the viral replication titer gradually increased with the advancement of the virus challenge time, with a latency period within 12 h after challenge, with little change in viral titer, after which,the virus starts to enter the logarithmic phase, the replication is faster between 12 and 24 hours, the replication titer reaches the highest value at 72 and h after the virus inoculation, the result shows that the ARV-HLJ21-1690401 strain can be well proliferated on LMH cells and has higher replication titer, and the replication titer can reach 10 at 72 and h after infection 7.0 TCID 50 and/mL or more.
2.6 Pathogenicity test for isolation of virus ARV-HLJ21-1690401
The ARV-HLJ21-1690401 strain is inoculated into SPF chick embryo, chick embryo death starts to appear on the 4 th day (4 dpi) after infection, the dead chick embryo is in whole body bleeding and dysplasia; by 7 dpi, chick embryo mortality was 90%, whereas the control group did not show chick embryo death throughout the observation period. After the ARV-HLJ21-1690401 strain was inoculated into SPF chicks of 6 weeks of age, 100% of infected chickens developed, and developed chickens exhibited clinical symptoms such as listlessness, anorexia, and paw pad swelling (FIG. 6), and the control group did not develop chicken development and paw pad swelling during the whole test period.
2.7 Cross neutralization assay for isolation of virus ARV-HLJ21-1690401
Cross neutralization experiments showed that ARV-HLJ21-1690401 virus could be neutralized by isotype positive serum but not other genotypic positive serum, indicating a lower cross neutralization reaction between this strain and other genotypic strains, with greater antigenic differences between this strain and other genotypic strains (Table 2).
Example 2 preparation of inactivated vaccine and study of its immunopotency
1. Method of
1.1 Preparation of inactivated vaccine
The isolated virus ARV-HLJ21-1690401 of example 1.1 was inoculated into chick embryo fibroblasts at a ratio of 10% V/V, cultured to 84 h, and when the cytopathic effect reached 80% or more, the virus was collected, designated as F1 generation, and serial passage was performed on chick embryo fibroblasts to F6 generation in the same manner, the titer was measured, and adjusted to 10 6.0 TCID 50 0.1mL. Adding formaldehyde with a volume ratio of 1 per mill into F6 generation virus culture solution with a good titer, uniformly mixing, placing at 37 ℃, rotating and uniformly mixing for 1 time every 1 hour, and placing for 24 hours. Tween-80 with the volume ratio of 5% V/V and inactivated virus liquid is 1:20, and emulsifying with white oil according to the volume ratio of 1:1.5. The specific operation of emulsification is as follows: adding white oil into an emulsifying bottle to emulsify for 30s, and slowly adding the virus mixed solution along an emulsifying head, wherein no bubbles are generated. Regulating the emulsifying device to 6 grades, emulsifying for 3 min/time, repeating for three times, and dripping the emulsified sample into water, wherein the emulsified sample is not scattered for 5-10 s.
1.2 Safety test of inactivated vaccine
10 SPF chickens of 7 days old are intramuscular injected with 1.0ml of the inactivated vaccine prepared in 1.1 per leg, 5 control groups are simultaneously provided, the control groups are inoculated with the same volume of emulsifying adjuvant without virus by the same way, the chickens are fed under the same condition, the continuous observation is carried out for 14 days, and the feeding, drinking and clinical conditions of the experimental chickens are observed.
1.3 Efficacy test of inactivated vaccine
The inactivated vaccine prepared in 1.1 was inoculated into 10 SPF chickens of 3 weeks of age by intramuscular injection through legs at a dose of 1.0 ml/dose, collected from 1w,2w,3w after immunization and serum was isolated, and antibody levels were detected. At the same time, 5 non-immunized control groups were set.
1.4 Toxicity attack protection test of inactivated vaccine
10 SPF chickens of 7 days old were given 1.0ml of the inactivated vaccine prepared in 1.1 intramuscular injection to each leg, while 5 control animals were fed under the same conditions. All SPF chickens were tested for protection against challenge with ARV-HLJ21-1690401 F6-generation isolate virus at 3w post immunization, at a challenge dose of 10 4.5 TCID 50 The inoculation mode is single-side claw-pad inoculation, namely, the left side claw-pad inoculation is performed on each chicken, the right side claw-pad is used as a self-control, and the non-immune control group attacks the same dose of virus liquid in the same mode. And continuously observing for 10 days after the virus attack, observing and recording the morbidity of the SPF chicken every day, and counting the protection rate.
2. Results
2.1 Safety test of inactivated vaccine
The results show that the chickens in the test group and the control group only develop normally, have good mental state, and the injection site vaccine is found to be absorbed well by the section test group, so that the inflammatory reactions such as red swelling, tissue necrosis and the like are avoided. The inactivated vaccine prepared by the method is safe and harmless, and has no influence on animal growth.
2.2 Efficacy test of inactivated vaccine
The strain is inactivated and emulsified to immunize SPF chicken to induce better antibody level, the antibody level gradually rises along with the advancement of immunization time, and the average antibody level can reach 2800 or more after 3 weeks of immunization (figure 7); the result shows that the strain has good immunogenicity, can be used for preparing inactivated vaccines, and provides a basis for comprehensive prevention and control of the disease in poultry farms in China.
2.3 Toxicity attack protection test of inactivated vaccine
ARV-HLJ21-1690401 strain is prepared into an inactivated vaccine to immunize SPF chicken, the chicken is continuously observed for 10 days after virus challenge, and the clinical symptoms of the chicken are observed every day and the morbidity is recorded. The patients with symptoms such as redness and swelling of paw pad, joint swelling, lameness, depression of spirit, and appetite decrease, and symptoms of the same chicken occurring continuously for 5 days, are marked as morbidity. In the whole observation period of the inactivated vaccine immune group, the chickens only do not have any disease state, all the chickens have good mental state, and the claw cushion does not have any swelling state; the virus-attack control group starts to attack after 2 days of virus attack, the attack lasts for the whole observation period, and the attack chicken shows clinical symptoms such as listlessness, anorexia, serious swelling of the paw pad and the like.
The results show that the inactivated vaccine prepared by the ARV-HLJ21-1690401 strain can provide complete protection for the isogenic virulent strain, and the strain is expected to be used as a candidate vaccine strain for the reserve research of the current novel genotype vaccine of the chicken reovirus.
Claims (5)
1. A variant strain of the Avian reovirus type III (ARV) is named ARV-HLJ21-1690401 and is preserved in the China general microbiological culture Collection center (China Committee for culture Collection), and has the preservation number: CGMCC No.45563.
2. Use of a variant avian reovirus type III according to claim 1 for the preparation of a medicament for the prevention of avian reovirus disease.
3. The use of claim 2, wherein the medicament is an inactivated avian reovirus vaccine.
4. An inactivated vaccine for avian reovirus gene III, characterized in that the active ingredient of the inactivated vaccine is the inactivated variant of avian reovirus gene III as claimed in claim 1.
5. A method for preparing an inactivated vaccine for avian reovirus gene type III according to claim 4, comprising the steps of:
adding formaldehyde with a volume ratio of 1 per mill into the F6 generation virus culture solution of the gene III type avian reovirus variant strain of claim 1, uniformly mixing, placing at 37 ℃ for 1 time after every 1 hour of rotation, and placing for 24 hours to obtain an inactivated virus solution; tween-80 with the volume ratio of 5% V/V and inactivated virus liquid is 1:20, mixing uniformly, and emulsifying with white oil according to the volume ratio of 1:1.5, wherein the specific operation of emulsification is as follows: adding white oil into an emulsifying bottle to emulsify for 30s, slowly adding virus mixed solution along an emulsifying head, taking care of not generating bubbles, regulating an emulsifying device to 6 grades, emulsifying for 3 min/time, repeating for three times, dripping the emulsified sample into water, and enabling the emulsified drops not to scatter for 5-10 s.
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