Duck plague virus QL2312 strain and application thereof in preparation of duck plague virus inactivated vaccine

Abstract

The invention provides a duck plague virus QL2312, and further provides application of the duck plague virus QL2312 in preparation of a duck plague virus infected animal model and application of the duck plague virus QL2312 in preparation of vaccines for preventing duck plague virus infected diseases. The duck plague virus QL2312 strain attacks 2-12 month-old susceptible ducks, and the death rate of the infected ducks reaches 100%. The QL2312 strain is inactivated by formaldehyde and then emulsified with an oil adjuvant to prepare an inactivated vaccine for immunizing test ducks, and the duck plague virulent AV1221 strain is used for challenging to verify the protection capability of the inactivated vaccine. Experimental results show that the survival rate of 2-12-month-old ducks immunized by the inactivated vaccine is kept consistent with that of duck plague live vaccines, and the survival rate of 1-21-day-old ducks immunized by the inactivated vaccine is remarkably superior to that of commercially available duck plague live vaccines. Therefore, the separated and purified QL2312 strain can be used for preparing inactivated vaccines and can effectively prevent death caused by duck plague infection of ducks of different day ages.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of virus isolation and vaccine preparation, in particular to a duck plague virus, and further relates to an inactivated vaccine prepared from the duck plague virus. BACKGROUND

[0002] Duck Plaque Virus (DPV), also known as Duck virus Enteritis Virus (DEV), is a pathogen of the disease of acute onset, septicemia and high mortality infectious disease with fast transmission speed, which is caused by the duck of the order Anseriformes and the family Anatidae. The duck plague virus is a member of the duck alphaherpesvirus 1 of the Mardivirus genus of the alphaherpesvirinae subfamily of the herpesviridae family, and the genomic structure of the duck plague virus is similar to that of other herpesviruses. Both of them are UL-IRS-US-TRS, i.e. Unique long (UL), Inverted repeat short (IRs), Unique short (US) and Terminal repeat short (TRs). The genome length is 158-162 kb, the G+C content is 44.9%, it is a double-stranded linear DNA, and it encodes 78 open reading frames (ORF).

[0003] Duck plague virus can infect different breeds and different ages of ducks, and is one of the main pathogenic agents of duck industry. In natural conditions, growing ducks and adult ducks are more likely to get sick, but in recent years, the prevalence of duck plague has shown some new changes. Ducklings of 10-15 days old have also been infected with duck plague, and immune duck groups have also shown typical and atypical cases of duck plague. In 2016, duck groups on egg duck farms in Zhejiang Province were infected with duck plague vaccine, and Zhang Hongli et al. analyzed the ZJ2016 strain and found that LORF11, UL2, UL12, UL41, UL47, and US10 genes were consistent with the characteristics of virulent strains. Compared with the vaccine strain, the LORF11, UL2, UL12, and US10 genes had base insertions, resulting in changes in gene structure and amino acids. Fu Guanghua et al. analyzed 37 duck plague samples from different regions of Fujian in 2016-2017 and found that the UL56 / LORF5 gene, TK / gH gene, and UL2 gene did not have gene fragment deletions, and the sequence similarity with the reference virulent strain in China was more than 99%. However, molecular phylogenetic analysis found that the strain was in a different evolutionary branch from the reference virulent strain in China. Zhang Rui et al. isolated a virulent strain of duck plague virus, FJ2020176, from half-breed ducks in Fujian Province in 2020. The UL2 gene of this strain had a nucleotide homology of 97.8%-99% with the duck plague virulent strains CHv, CV, and 2085. Compared with the attenuated strains VAC, Attenuated strain 1, and Attenuated strain 2, there was a 528bp nucleotide insertion in the UL2 gene. Tang et al. isolated a virulent strain of duck plague, SD423, from 10-20 day old duck groups that died in the spring of 2023. The genomic sequence had a nucleotide similarity of 99.90% and an amino acid similarity of 99.75% with the Chinese virulent strain SD (MN518864.1). The UL2 gene had the highest homology with the SD and 2028 strains, and the UL56 / LORF11 gene had the highest homology with the SD strain.

[0004] Currently, 2-3 times of duck plague vaccine are used for prevention and control before laying ducks, resulting in high maternal antibody levels in ducklings, long duration, poor passive immune protection, and interference of maternal antibodies with duck plague live vaccine immunization, which seriously affects the prevention and control effect of duck plague in live vaccine immunized ducklings. In view of the problem that duck plague live vaccine cannot avoid the interference of maternal antibodies, a duck plague variant strain is isolated from sick ducks in the present application. Genome analysis shows differences from the classical virulent strain AV1221, the vaccine strain K-CKE, and other isolated variant strains, and the titer after inoculation into chicken embryo fibroblasts is higher than that of other isolated strains. Immunization of 1-week-old ducklings with the duck plague inactivated vaccine prepared from the strain can achieve a 80% protection rate after 10 days of challenge and a 100% protection rate after 21 days of challenge. Compared with other inactivated vaccines, the protective immune time is earlier, the protection effect is good, and it can be used for 1-day-old ducklings to prevent and control duck plague. SUMMARY

[0005] The present application aims to overcome the defects of the prior art, and provides a newly isolated duck plague virus, and verifies that the inactivated vaccine prepared from the virus can produce better protection against infection in immunized ducks.

[0006] Based on this, the present application provides a duck plague virus (Duck Plaque Virus, DPV) QL2312 strain, which was deposited with the China General Microbiological Culture Collection Center on May 23, 2025, and the deposit number is CGMCC No. 45409.

[0007] The present application also provides the application of the above-mentioned duck plague virus (Duck Plaque Virus, DPV) QL2312 strain in preparing a duck plague virus-infected animal model.

[0008] Preferably, the challenge dose for preparing the animal model is 1.0 ml / bird, and the virus content is 10 6.0 TCID 50 , and the challenge method is leg muscle injection.

[0009] On this basis, the present application also provides the application of the above-mentioned duck plague virus (Duck Plaque Virus, DPV) QL2312 strain in preparing a drug for preventing duck plague virus infection.

[0010] In particular, the present application provides a duck plague virus inactivated vaccine, which contains the above-mentioned duck plague virus (Duck Plaque Virus, DPV) QL2312 strain.

[0011] In the present application, the duck plague virus (Duck Plaque Virus, DPV) QL2312 is inactivated, and the virus titer before inactivation is not less than 10 8.0 TCID 50 / ml.

[0012] On the other hand, the present application also provides a preparation method of the above-mentioned duck plague virus vaccine, which comprises the following steps:

[0013] (1) inoculate the duck plague virus (Duck Plaque Virus, DPV) QL2312 strain into CEF cells, collect the CEF cell virus liquid, and then add 4% sterilized Tween-80 by total volume percentage after inactivation, and fully stir until the Tween-80 is completely dissolved to obtain an aqueous solution;

[0014] (2) in a sterile beaker, add the oil phase solution, start the emulsifier, then slowly add the water phase until the volume ratio of the oil phase solution to the water phase solution is 2:1, fully emulsify under stirring to obtain the inactivated vaccine of duck plague virus QL2312 strain;

[0015] The oil phase solution is prepared by adding 1g of aluminum stearate into 188ml of mineral oil, slowly heating to 80℃ and fully stirring until completely dissolved, then adding 12ml of Span-80, uniformly stirring and continuously heating to 115℃ under fully stirring, and cooling to obtain.

[0016] Preferably, the duck plague virus QL2312 strain is inoculated with CEF cells that grow well into a single layer, and the inoculation amount is 1% of the culture solution, and after mixing, the CEF cell culture is placed in a 37℃, 5% CO2 incubator for continuous culture, and when more than 80% of the CEF cells show lesions, the CEF cell culture is harvested as the virus solution.

[0017] In addition, the application also provides the application of duck plague virus (DPV) QL2312 strain in detecting the efficacy of duck plague virus vaccine.

[0018] As a preferred embodiment, 10 healthy susceptible ducks aged 2-12 months are used, and 5 of them are in the immunization group and the other 5 are in the control group, the ducks in the immunization group are subcutaneously injected with 0.5ml of the vaccine on the back of the neck, and the ducks in the control group are not treated, 21-28 days later, each duck is injected with 1.0ml of duck plague virulent strain AV1221 (containing 10 3 MLD) in the muscle, and 5 / 5 ducks in the control group die within 5 days after the attack, and none of the ducks in the immunization group dies within the 14-day observation period, and all the ducks are healthy.

[0019] The duck plague virus strain is isolated and identified from a certain duck farm in Jiangsu, the pathogenicity and genomic characteristics of the strain are analyzed, the molecular characteristics of the newly prevalent duck plague in duck flocks in Jiangsu province in recent years are determined, and the inactivated vaccine is prepared by using the prevalent strain.

[0020] The experiment proves that the duck plague virus QL2312 strain attacks 2-12-month-old susceptible ducks, and the mortality rate of the infected ducks reaches 100%, which is used as an animal model. The QL2312 strain is inactivated by formaldehyde and emulsified with an oil adjuvant to prepare an inactivated vaccine for immunizing the test ducks, and the virulent duck plague strain AV1221 is used for attack to verify the protection ability of the inactivated vaccine. The experimental results show that the survival rate of the 2-12-month-old ducks immunized by the inactivated vaccine of the application is consistent with that of the duck plague live vaccine, and the survival rate of the 1-21-day-old ducks immunized by the inactivated vaccine of the application is significantly better than that of the commercially available duck plague live vaccine. Therefore, the isolated and purified QL2312 strain can be used for preparing the inactivated vaccine, and can effectively prevent the death of ducks of different ages caused by duck plague infection.

[0021] Deposit information

[0022] Duck Plaque Virus QL2312, preserved in China General Microbiological Culture Collection Center on May 23, 2025, with the preservation number of CGMCC No. 45409. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Identification of anatomical lesions of the diseased duck in the sample of Example 1.

[0024] Figure 2 PCR identification of clinical samples.

[0025] Figure 3 Growth characteristics of Duck Plaque Virus QL2312 strain on DEF cells.

[0026] Figure 4 Kaplan-Meier survival curves of ducks infected with AV1221 strain and QL2312 strain, respectively.

[0027] Figure 5 Anatomical lesion map of Duck Plaque Virus animal regression test.

[0028] Figure 6 Phylogenetic tree of Duck Plaque Virus whole genome sequence.

[0029] Figure 7 Genome structure map of Duck Plaque Virus. DETAILED DESCRIPTION

[0030] The following examples are used to illustrate the technical solutions of the present application, but do not limit the scope of the present application.

[0031] In the present application, unless otherwise specified, "%" used to explain the concentration is volume percentage, and ":" is weight ratio.

[0032] In the following examples, all experimental methods used are conventional methods, or are carried out according to the instructions of the kit. The materials, reagents or instrument equipment used in the present application are not marked with the manufacturer, and are conventional products that can be purchased on the market.

[0033] Example 1 Isolation and identification of virus

[0034] (1) The diseased duck sent by a duck farm in Jiangsu Province has typical duck plague lesions, such as depression, head and neck edema, tearing, and eye and face edema. The liver is found to be hemorrhagic and enlarged, the thymus is hemorrhagic, the neck subcutaneous tissue is edematous, the trachea is ring hemorrhagic, the intestinal tract is ring hemorrhagic, and the color is deep red or dark brown, and the bursa of Fabricius has hemorrhagic spots, as shown in Figure 1 .

[0035] (2) PCR detection

[0036] In sterile environment, liver tissue of dead duck was picked up and inoculated on different culture medium plates, which were placed in 37℃ incubator for 48h. No colony growth was observed on the inoculated plates, which could exclude bacterial infection. The clinical sample swabs, liver, spleen, thymus, trachea, esophagus and bursa of Fabricius were respectively identified for duck plague virus. In addition, the samples were mixed and detected for duck common pathogens such as avian influenza virus, Newcastle disease virus and Tembusu virus.

[0037] Among them, the duck plague virus identification primer was synthesized artificially:

[0038] DPV-F: 5'-GCAAGCTTGGCTGGTATGCGTGACAT-3'

[0039] DPV-R: 5'-TTCTGCAGGTATTGGTTTCTGAGTTGGC-3'

[0040] According to the operation steps of the virus nucleic acid extraction kit (MagaBio plus virus DNA / RNA purification kit III) instruction, the nucleic acid (DNA and RNA) was extracted, and the sample was identified according to the one-step RT-PCR kit HiScript IIOne Step RT-PCR Kit (Dye Plus) instruction. Reaction system: RNase-free ddH2O 16.5μl, 2×One Step Mix (Dye Plus) 25μl, One Step Enzyme Mix 2.5μl, Primer F (10μM) 2μl, Primer R (10μM) 2μl, template DNA 2μl; 50℃ 30min, 94℃ 3min, 94℃ 30s, 56℃ 30s, 72℃ 1min, 30 cycles, 72℃ 7min. The PCR product was detected by 0.8% agarose gel electrophoresis.

[0041] The results are shown in Figure 2 The PCR product band was about 602bp in liver, spleen and trachea tissues, confirming that duck plague was detected in the sample, and the rest of the pathogens were negative.

[0042] (3) Inoculation of duck embryo fibroblasts

[0043] The supernatant of duck plague virus positive liver sample was inoculated into well-grown monolayer duck embryo fibroblasts (DEF cells) culture, which was blind passed for 3 generations. Under the microscope, DEF cell lesions were observed, which showed cell netting, vacuoles in cytoplasm, and the lesion range gradually expanded, shed and died over time, as shown in B of Figure 3 Figure 3 ​A is control). The genome was extracted and detected by PCR to be positive for DV, as shown in Figure 3 The obtained strain was named as QL2312 strain.

[0044] (4) Determination of virus growth curve

[0045] QL2312 strain was inoculated into DEF cells with a 1% inoculation dose, and the virus was harvested at 12h, 24h, 36h, 48h and 60h after inoculation. The virus liquid was serially diluted by 10 times in a sterile 1.5ml centrifuge tube, 0.1ml of the diluted virus liquid was taken and inoculated into DEF cells in a 96-well cell culture plate, 8 wells were inoculated with each dilution of the virus liquid, and the plate was incubated in a 37°C cell culture incubator containing 5% CO2. The plate was observed until 120h after inoculation, the pathological changes of each well were recorded, and the TCID 50 of the virus was calculated according to the Reed-Muench method.

[0046] It was found that the virus titer of QL2312 strain rapidly increased at 12-36h after inoculation, the virus titer slowly increased at 36-48h, reached the maximum value of 10 7.5 TCID 50 / ml at 48h, and then slowly decreased, as shown in Fig. D. Figure 3

[0047] (5) Animal regression test

[0048] Thirty 4-week-old SPF ducklings were randomly divided into three groups, 10 ducklings per group. The ducklings in the first group were inoculated with DV QL2312 strain by intramuscular injection in the leg, 1.0ml per duckling (containing 10 6.0 TCID 50 ), the ducklings in the second group were inoculated with DV virulent strain AV1221 by intramuscular injection in the leg, 1.0ml per duckling (containing 10 3.0 MLD), and the ducklings in the third group were not inoculated as control. After inoculation, the ducklings were isolated and fed, and the clinical manifestations and incidence of the ducklings were observed and recorded daily for 14d. The trachea, liver, kidney and bursa of Fabricius were dissected and observed for pathological changes.

[0049] The results showed that the ducklings in the artificial infection group appeared symptoms such as depression, slow walking and lying down 3d after inoculation with DV QL2312 strain, and 4 ducklings died 4d after inoculation, all the ducklings died 6d after inoculation, and the morbidity and mortality were both 100%, as shown in Fig. C. The pathological changes of the organs of the dead ducklings were consistent with those of the clinical natural infection, and the organs were damaged to different degrees, as shown in Fig. D. Figure 4 Figure 5

[0050] ​​​Therefore, the digestive system, immune system and urinary system of SPF ducklings infected with the duck plague virus of the application are damaged.

[0051] (6) Genetic evolution analysis of duck plague virus

[0052] The purified nucleic acid DNA of the duck plague virus QL2312 strain was extracted according to the operation steps of the virus nucleic acid extraction kit (MagaBio plus virus DNA / RNA purification kit III) and mailed to Shanghai Shengong Engineering Co., Ltd. for whole genome sequencing.

[0053] The software was used for virus homology analysis and genetic evolution phylogenetic tree drawing. The nucleotide homology analysis of QL2312 strain and the representative strains of duck plague published in GenBank was performed, and the phylogenetic tree was constructed to test the reliability of the phylogenetic tree, and the repeated analysis was performed for 1000 times.

[0054] The whole genome sequencing genome is 162128 bp in length, and the GC content is 44.6%. A total of 78 open reading frames are encoded, and the nucleotide similarity with the classical virulent strains CHv strain (GenBank accession number JQ647509), CV strain (GenBank accession number JQ673560), 2085 strain (GenBank accession number NC075687) and SD strain (GenBank accession number MN518864) is 99.71%, 99.70%, 93.33% and 98.85%, respectively; and the nucleotide similarity with the chicken embryo vaccine strain C-KCE strain (GenBank accession number KF263690) is 97.37% (Table 1).

[0055] Table 1 Homology analysis of six duck plague virus strains

[0056]

[0057] The homology analysis results of the six strains show that the nucleotide similarity of QL2312 strain with the classical virulent strains CHv strain and CV strain is the highest. The phylogenetic tree analysis of the whole genome of the six duck plague virus strains shows that QL2312 strain has a close genetic relationship with the chicken embryo vaccine strain C-KCE strain, as shown in Figure 6 .

[0058] Among them, the nucleotide and amino acid sequences of UL2, UL56 and LORF5 obtained by sequencing of QL2312 strain are shown as SEQ ID No. 1-6; the nucleotide and amino acid sequences of LORF11 of the chicken embryo vaccine strain C-KCE strain are shown as No. 7-8

[0059] (7) Analysis of virus UL56 / LORF5 and UL2 genes

[0060] Since the existing vaccine strains in China have gene deletion in UL56 / LORF5 and UL2 genes, the vaccine attenuated strain and the virulent strain can be distinguished. The primers are synthesized by Shanghai Shengong Bioengineering Co., Ltd. and the primer sequences are shown in Table 2.

[0061] Table 2 Amplification primers used in the experiment

[0062]

[0063] The nucleic acid (DNA and RNA) is extracted according to the operation steps of the virus nucleic acid extraction kit (MagaBio plus virus DNA / RNA purification kit III) and the sample is identified according to the 2x Taq Master Mix (Dye Plus) kit instructions. The amplification primers are shown in Table 2 and the amplification reaction system is 50 μl: 2 μl of upstream and downstream primers, 2 μl of DNA, 25 μl of 2x Prime STAR Max, and water to 50 μl. The reaction program is 98℃ for 5 min; 98℃ for 15 s; 53℃ for 15 s; 72℃ for 50 s; 30 cycles; 72℃ for 10 min. The PCR product is detected by 0.8% agarose gel electrophoresis.

[0064] The results show that the PCR fragment of 4603 bp obtained by amplifying the UL56 / LORF5 gene of QL2312 strain is highly similar to the homologous passage strain sequence of the virulent strain CHv and CV isolated from duck flocks in China, and the nucleotide similarity is between 99.8-100%. The difference is that the vaccine attenuated strain C-KCE has a continuous deletion of 3513 bp nucleotides at the UL56 / LORF5 position, and the amplified PCR fragment is 1090 bp. The results prove that the UL56 / LORF5 gene of the duck plague virus QL2312 strain of the application is consistent with the sequence of the virulent strain in China, and there is no nucleotide deletion or insertion, as shown in Fig. B. Figure 7

[0065] In addition, the duck plague virus UL2 is a homologous gene of UL2 of Herpes simplex virus type 1 (HSV-1), which encodes uracil DNA glycosylase, which is an enzyme that plays an important role in virus replication, and its influence on virulence needs further study. The UL2 gene of QL2312 strain is amplified to obtain a 1311 bp fragment containing the whole UL2 gene, which is the same as the amplified fragment of the vaccine attenuated strain C-KCE. Compared with the virulent strain CV, the newly isolated duck plague virus QL2312 strain has a 528 bp nucleotide deletion in the UL2 gene, which is consistent with the sequence of the attenuated strain in China, as shown in Fig. C. Figure 7 ​The results show that the duck plague virus QL2312 strain has the characteristics of both a strong strain (UL56 / LORF5 gene is not deleted) and a weak vaccine strain (UL2 gene is deleted), which is different from other existing vaccine strains.

[0066] Example 2 Preparation and test standards of inactivated vaccine

[0067] (1) Preparation of CEF monolayer cells

[0068] According to the current "Chinese Veterinary Pharmacopoeia" Appendix, CEF is prepared, and a cell suspension containing about 1 million to 1.5 million live cells per 1.0 ml is prepared and divided into 3000 ml culture bottles. The culture is incubated at 37°C in a warm room at 8-10 revolutions per hour, and a good monolayer is formed for use.

[0069] (2) Inoculation of virus

[0070] When the CEF cells grow into a good monolayer, inoculate the duck plague virus QL2312 strain at a dose of 1% of the culture volume. Mix well and continue to culture at 37°C in a warm room at 8-10 revolutions per hour. Observe the cells once a day. When more than 80% of the CEF cells show pathological changes, store the culture bottles at -15°C or below.

[0071] (3) Virus content determination

[0072] CEF cell culture is diluted 10 times in a series of dilutions with serum-free maintenance fluid. Take 10 -5 , 10 -6 , 10 -7 , 10 -8 4 dilutions, inoculate the CEF cells that have grown into a good monolayer in 96-well cell culture plates, 8 wells for each dilution, 100 μl per well, and set up non-inoculated control wells at the same time. Incubate in a 37°C, 5% CO2 incubator, observe for 120 hours, and count the number of CPE wells for each dilution. Calculate the TCID 50 according to the Reed-Muench method.

[0073] (4) Virus inactivation

[0074] CEF cell culture virus with a titer of 10 8.0 TCID 50 / ml or more is poured into an inactivation bottle, and 10% formaldehyde solution is added to mix thoroughly until the final concentration of formaldehyde solution is 0.2%. Then place it in a 37°C shaker for 48 hours of inactivation. The inactivated virus solution is stored at 2-8°C.

[0075] (5) Sterility test

[0076] According to the current "Chinese Veterinary Pharmacopoeia" Appendix.

[0077] (6) Inactivation test

[0078] The inactivated duck plague virus solution was diluted 10 times, and inoculated into CEF cells in a 6-well cell culture plate according to the maintenance solution 2% volume inoculation. CEF cell blank control was set up, and observed for 5 days. If no cytopathic effect appeared in the cell culture plate, the culture was harvested and repeatedly frozen and thawed 3 times, and then passaged blindly for 1 generation. The CEF cells were inoculated again, and observed for 5 days continuously to confirm whether the cells produced cytopathic effect.

[0079] (7) Emulsification

[0080] Mineral oil 188 ml was measured, and 1 g of aluminum stearate was added. The mixture was slowly heated to 80°C, and stirred at 200 r / min to dissolve the aluminum stearate. Then, 12 ml of Span-80 was added, and stirred uniformly. The mixture was continuously heated to 115°C and maintained for 30 minutes. After uniform stirring, the mixture was cooled and prepared for use.

[0081] The inactivated qualified duck plague virus antigen 96 ml was measured, and 4 ml of sterilized Tween-80 was added. The mixture was stirred thoroughly to completely dissolve the Tween-80. The oil phase was placed in a sterile beaker, and the emulsifying machine was started. The water phase was slowly added, and the speed was increased to 25000 r / min to continue emulsification for 3 minutes. After emulsification, 10 ml of the vaccine was measured and added into a centrifuge tube. The tube was centrifuged at 3000 r / min for 30 min, and the water phase separated from the bottom of the tube should not exceed 0.5 ml. After emulsification, the QL2312 strain inactivated vaccine was obtained.

[0082] (8) Safety test

[0083] Ten 10-14 day-old healthy susceptible ducks were used, and each duck was subcutaneously injected with 1.0 ml of inactivated vaccine on the neck back. The ducks were observed for 14 days, and all the ducks should be healthy and no local or systemic adverse reactions caused by the vaccine should occur.

[0084] (9) Potency test

[0085] Five 2-12 month-old healthy susceptible ducks were used, and each duck was subcutaneously injected with 0.5 ml of the vaccine on the neck back. Twenty-one to twenty-eight days later, five control ducks were used, and each control duck was intramuscularly injected with 1.0 ml of duck plague virulent strain AV1221 (containing 10 3 MLD). The ducks were observed for 14 days after the challenge, and all the control ducks should be sick and dead. At least four of the immune ducks should be healthy.

[0086] Example 3 Inactivated vaccine test and comparison of the potency of duck plague live vaccine

[0087] (1) Safety test with 10-day-old healthy susceptible ducks 10, each duck subcutaneously injected with inactivated vaccine 1.0ml in the neck back, observed for 14d, 10 / 10 healthy and alive, and no local or systemic adverse reactions caused by the vaccine. It shows that the inactivated vaccine prepared by the method is qualified for safety test.

[0088] (2) Efficacy test with 2-12 month-old healthy susceptible ducks 5, each subcutaneously injected with vaccine 0.5ml in the neck back, 21-28 days later, together with control ducks 5, each injected with duck plague virulent strain AV1221 1.0ml (containing 10 3 MLD) in the muscle. The control group died within 5d after challenge, and the immune group did not die within 14d of observation, and all were healthy and alive. The specific death is shown in Table 3.

[0089] Table 3 Observation record after challenge in efficacy test

[0090]

[0091]

[0092] The above results show that the inactivated vaccine prepared by the duck plague virus QL2312 strain of the application is qualified for efficacy test.

[0093] (3) Application of duck plague inactivated vaccine

[0094] (3.1) Take 1-day-old healthy cherry valley meat ducks 20, divided into 2 groups, group 1 subcutaneously injected with QL2312 strain inactivated vaccine 0.5ml containing 1 feather, group 2 subcutaneously injected with commercially available duck plague live vaccine 0.5ml containing 1 feather, and set up control group 5 under the same conditions. 21 days after immunization, all test ducks were injected with duck plague virulent strain AV1221 1.0ml (containing 10 3 MLD) in the muscle. 6d after challenge, 5 / 5 of the control group died, 10 / 10 of the commercially available duck plague live vaccine immune group died, and 2 / 10 of the duck plague QL2312 strain inactivated vaccine immune group died. The specific death is shown in Table 4.

[0095] Table 4 Observation record after challenge in 1-day-old healthy cherry valley meat ducks

[0096]

[0097] (3.2) Take 21-day-old healthy cherry valley meat ducks 20, divided into 2 groups, group 1 subcutaneously injected with QL2312 strain inactivated vaccine 0.5ml containing 1 feather, group 2 subcutaneously injected with commercially available duck plague live vaccine 0.5ml containing 1 feather, and set up control ducks 5 under the same conditions. 21 days after immunization, all test ducks were injected with duck plague virulent strain AV1221 1.0ml (containing 10 3The control group had 5 / 5 deaths, and the commercial duck plague live vaccine immunized group and the duck plague QL2312 strain inactivated vaccine immunized group had 10 / 10 healthy ducks within 14 days of observation. Table 5 shows the death records of 21-day-old healthy cherry valley meat ducks after challenge.

[0098] Table 5: Observation records of 21-day-old healthy cherry valley meat ducks after challenge

[0099]

[0100] (3.3) 20 six-month-old healthy susceptible cherry valley meat ducks were divided into two groups. Group 1 was injected subcutaneously on the neck and back with QL2312 strain inactivated vaccine 0.5 ml containing 1 dose, and group 2 was injected subcutaneously on the neck and back with a commercially available duck plague live vaccine 0.5 ml containing 1 dose. A control group of 5 ducks was set up under the same conditions. After 21 days of immunization, all test ducks were injected intramuscularly with duck plague virulent strain AV1221 1.0 ml (containing 10 3 The control group had 5 / 5 deaths, and the commercial duck plague live vaccine immunized group and the duck plague QL2312 strain inactivated vaccine immunized group had 10 / 10 healthy ducks within 14 days of observation. Table 5 shows the death records of 21-day-old healthy cherry valley meat ducks after challenge.

[0101] Table 6: Observation records of 6-month-old healthy cherry valley meat ducks after challenge

[0102]

[0103] In summary, the duck plague virus strain QL2312 was isolated and identified from a duck farm in Jiangsu Province. The pathogenicity and genomic characteristics of the strain were analyzed, and the molecular characteristics of the newly prevalent duck plague virus in duck flocks in Jiangsu Province in recent years were determined. The QL2312 strain has the characteristics of a virulent strain (UL56 / LORF5 gene is not deleted) and a weak vaccine strain (UL2 gene is deleted).

[0104] The inactivated vaccine prepared using the prevalent strain was tested on animals, and the results showed that the duck mortality rate reached 100% after infection with the duck plague virus QL2312 strain in 2-12 month-old susceptible ducks, which was used as an animal model. The QL2312 strain was inoculated into CEF cells, inactivated by formaldehyde, and emulsified with oil adjuvant to prepare an inactivated vaccine for immunization of test ducks. The duck plague virulent strain AV1221 was used for challenge to verify the protection ability of the inactivated vaccine. The test results showed that the survival rate of 2-12 month-old ducks immunized with the inactivated vaccine of the application was consistent with that of the duck plague live vaccine, but the survival rate of 1-21 day-old ducks immunized with the inactivated vaccine of the application was significantly better than that of the commercially available duck plague live vaccine.

[0105] Therefore, the isolated QL2312 strain can be used to prepare an inactivated vaccine to effectively prevent death caused by duck plague infection in ducks of different ages.

Claims

1. A Duck Plaque Virus (DPV) strain QL2312, which was deposited with the China General Microbiological Culture Collection Center on May 23, 2025, and has a deposit number of CGMCC No.45409. 2.The use of the Duck Plaque Virus (DPV) strain QL2312 of claim 1 in the preparation of a DPV-infected animal model.

3. Use according to claim 2, characterized in that The dose for preparing the animal model is 1.0 ml / bird, and the virus titer is 10 6.0 TCID 50 / mL, and the infection mode is leg muscle injection. 4.The use of the Duck Plaque Virus (DPV) strain QL2312 of claim 1 in the preparation of a vaccine for preventing DPV infection diseases. 5.A DPV vaccine, which contains the Duck Plaque Virus (DPV) strain QL2312 of claim 1.

6. The duck plague virus vaccine according to claim 5, characterized in that The vaccine is an inactivated vaccine, and the duck plague virus titer before inactivation is not less than 10 8.0 TCID 50 / ml. 7.A method for preparing the DPV vaccine of claim 6, which comprises the following steps: (1) inoculating the Duck Plaque Virus (DPV) strain QL2312 into CEF cells, inactivating the virus liquid harvested from the CEF cells, then adding 4% sterilized Tween-80 in terms of the total volume percentage, and fully stirring until the Tween-80 is completely dissolved to obtain an aqueous phase solution; (2) adding an oil phase solution in a sterile beaker, then slowly adding the aqueous phase until the volume ratio of the oil phase solution to the aqueous phase solution is 2:1, fully emulsifying under stirring to obtain an inactivated vaccine of the Duck Plaque Virus QL2312 strain; the oil phase solution is prepared by adding 1 g of aluminum stearate into 188 ml of mineral oil, slowly heating to 80℃ and fully stirring until completely dissolved, then adding 12 ml of Span-80, stirring uniformly and continuing to heat to 115℃ under the condition of fully stirring to obtain uniformity, and cooling. 8.The use of the Duck Plaque Virus (DPV) strain QL2312 of claim 1 in the detection of the efficacy of a DPV vaccine.

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