Triple tetravalent inactivated vaccine for preventing and treating duck viral diseases and application of triple tetravalent inactivated vaccine

Abstract

The invention provides a triple tetravalent inactivated vaccine for preventing and treating duck viral diseases and application of the triple tetravalent inactivated vaccine, and belongs to the technical field of biological vaccines. A water phase of the vaccine is prepared by matching a type 1 duck hepatitis virus antigen solution, a type 3 duck hepatitis virus antigen solution and a duck reovirus antigen solution, and then adding a duck parvovirus VP2 protein solution to obtain a mixed antigen solution with the final concentration of 40 mu g / ml, the virus content in the type 1 duck hepatitis virus antigen solution and the virus content in the type 3 duck hepatitis virus antigen solution are 106.5 ELD50 / 0.2 ml, and the virus content in the duck reovirus antigen solution is 106.5 ELD50 / 0.2 ml. And the virus content of the duck reovirus antigen is 105.5 TCID50 / ml. The four antigen components in the vaccine are free of mutual interference, good in safety and exact in immune effect, the vaccine can achieve the purpose of preventing multiple diseases by one injection, the stress response of the duck immune vaccine is reduced, meanwhile, the four virus diseases are susceptible to small-day-age ducks, and invasion of the four viruses can be prevented at the same time after immunization. The vaccine preparation method is simple and convenient, and the production cost is greatly reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological vaccines, and particularly relates to a triple quadrivalent inactivated vaccine for preventing and treating duck viral diseases and its application. Background Art

[0002] Duck viral hepatitis is a highly contagious and fatal infectious disease caused by duck hepatitis virus. Duck viral hepatitis has an acute onset, rapid transmission speed, and high mortality rate. Clinically, it is characterized by rapid onset, convulsions, acute death, liver swelling and bleeding, and the most typical symptom is opisthotonus after death. The morbidity rate of ducklings can reach 100%. It can occur throughout the year, and ducklings within 3 weeks of age are the most susceptible. It is one of the most serious infectious diseases endangering the duck farming industry in China and can cause serious economic losses to the duck farming industry worldwide. Currently, there is no specific therapeutic drug, and the main prevention and control method is mainly quarantine monitoring. Currently, there are 3 serotypes of duck hepatitis virus, namely type I, type II, and type III. Among them, type I duck hepatitis virus has the strongest pathogenicity and is widely distributed worldwide. The 3 serotypes have obvious serological differences and no cross-immunity. Type I duck hepatitis virus is mainly prevalent in China, and cases of type III have been reported successively in recent years. For the prevention and treatment of duck hepatitis, at present, in addition to strengthening feeding management, vaccine immunization and targeted treatment play an important role in controlling the prevalence of the disease.

[0003] New duck reovirus (NDRV) disease is a newly emerging disease caused by reovirus, characterized by splenomegaly, bleeding, and necrosis. Since 2005, in Putian, Fuzhou, Changle, Fuqing, Zhangpu in Fujian Province, Foshan in Guangdong Province, and Zhejiang and other places in China, a disease mainly characterized by irregular necrosis and bleeding mixture of the liver, myocardial bleeding, splenomegaly with patchy necrosis, and bleeding of the kidneys and bursa of Fabricius has occurred in flocks of muscovy ducks, mule ducks, and mallards. This disease has no obvious seasonality and can occur in various breeds of ducks (such as muscovy ducks, mule ducks, mallards, Cherry Valley ducks, etc.), and there is an increasing trend year by year; the onset age is about 3 - 25 days old, with 5 - 10 days old being the majority. The course of the disease is 5 - 7 days, the morbidity rate is 5% - 20%, and the mortality rate is 2% - 15% (case fatality rate 40% - 75%). The younger the age or the more concurrent infections, the higher the morbidity and mortality rates, causing certain economic losses to the duck farming industry. Currently, there is no commercially available new duck reovirus-related vaccine, and many research institutes and biological product enterprises tend to study single vaccines and live vaccines for this disease.

[0004] The new duck parvovirus disease is a new disease with short beak and growth disorder as clinical features caused by the new duck-origin goose parvovirus or goose parvovirus variant. Since 2015, a new epidemic with short beak, exposed tongue, soft feet, poor feather growth, easy fracture, growth disorder and other clinical manifestations has broken out in Mule ducks and Cherry Valley ducks in Zhangzhou, Zhangpu, Longhai, Fujian Province and Shandong, Anhui, Jiangsu, Henan, Zhejiang and other provinces. The morbidity rate is 10% to 100%, the mortality rate is 2% to 10%, and the rate of dead duck elimination or crippled ducks is 20% to 80%, causing great economic losses to the duck industry. The disease is highly contagious and susceptible to young days. The incubation period is 5 to 7 days. It usually develops at 6 to 45 days of age. The course of the disease is long and lasts for more than 20 days. It occurs all year round. There is currently no commercially available vaccine for the new duck parvovirus disease. Most research institutions reported in the literature also tend to study the inactivated vaccine of the whole virus.

[0005] In recent years, my country's poultry industry has gradually developed from free-range farming to intensive farming, and the scale of farming has greatly increased compared with the past. New diseases and new outbreaks of old diseases of waterfowl have continued to appear, becoming a major problem that plagues farmers. Duck hepatitis, reovirus and duck parvovirus are all susceptible to ducks at a young age, and mixed infection or secondary infection of these three diseases is common in farms. There are currently no commercially available vaccines for the new duck reovirus and the new duck parvovirus, and many research institutes and biological product companies tend to study single vaccines or live vaccines for these two diseases. There are no reports of effective vaccines that can prevent and control these three diseases at the same time. Therefore, it is urgent to develop a triple quadrivalent inactivated vaccine for duck viral hepatitis (type 1 + type 3), new duck reovirus disease, and new duck parvovirus disease with good safety and effective immune effect. Summary of the invention

[0006] The present invention provides a triple quadrivalent inactivated vaccine for preventing and treating duck viral diseases and application thereof. The vaccine can not only achieve the purpose of multiple protections with one shot, but also reduce the stress response of ducks due to immunization with the vaccine. Meanwhile, young ducks are susceptible to the four viral diseases, and after immunization, the invasion of the four viruses can be prevented at the same time.

[0007] In order to achieve the above object, the present invention provides a triple quadrivalent inactivated vaccine for preventing and treating duck viral diseases, wherein the aqueous phase is prepared by mixing duck hepatitis virus type 1 antigen liquid, duck hepatitis virus type 3 antigen liquid and duck reovirus antigen liquid, and then adding duck parvovirus VP2 protein liquid to obtain a mixed antigen liquid with a final concentration of 40 μg / ml, wherein the virus content in the duck hepatitis virus type 1 antigen liquid and the duck hepatitis virus type 3 antigen liquid is 10 6.5 ELD 50 / 0.2ml, the virus content of duck reovirus antigen is 10 5.5 TCID 50 / ml.

[0008] Preferably, the volume ratio of the Duck Hepatitis virus type 1 antigen solution, Duck Hepatitis virus type 3 antigen solution and New Duck Reovirus antigen solution added to the aqueous phase is 1:1:1.

[0009] Preferably, the Duck Hepatitis virus type 1 strain is YBH 1 strain (Duck Hepatitis virus type 1), which was deposited at the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: V202513;

[0010] The Duck Hepatitis virus type 3 strain is YBH 3 strain (Duck Hepatitis virus type Ⅲ), which was deposited at the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: V202514;

[0011] The New Duck Reovirus strain is YBAH-03 strain (New Duck Reovirus), which was deposited at the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: V202511;

[0012] The recombinant baculovirus Re-Bac VP2 strain (Recombinant Baculovirus) expressing the VP2 protein of Duck Parvovirus was deposited at the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: V202512.

[0013] Preferably, the New Duck Reovirus is propagated by inoculating on the chicken liver cancer suspension cell LMH-C (Gallus gallus), and the virus content of the propagated New Duck Reovirus is 10 8.5 TCID 50 / 0.1ml. When preparing the vaccine, it is diluted to 10 5.5 TCID 50 / 0.1ml for use.

[0014] Preferably, the chicken liver cancer suspension cell LMH-C (Gallus gallus) was deposited at the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: C202558.

[0015] Preferably, the oil phase is obtained by uniformly mixing 96±2 parts of mineral oil and 0.67 part of aluminum stearate and heating to 80°C±5°C, then adding 4±2 parts of Span-80, maintaining at 115°C±5°C for 40±5 minutes, and cooling.

[0016] Preferably, when emulsifying the oil phase and the water phase, the volume ratio of the two added is 2:1. Among them, before emulsification, 96.6 ± 2 parts of the mixed antigen solution of the water phase and 3.4 ± 2 parts of sterile Tween-80 are fully mixed to completely dissolve Tween-80.

[0017] The present invention also provides an application of the triple-valent and tetravalent inactivated vaccine according to any one of the above technical solutions in the simultaneous prevention and treatment of duck hepatitis A virus type 1 disease, duck hepatitis A virus type 3 disease, novel duck reovirus disease, and novel duck parvovirus disease.

[0018] Preferably, healthy and susceptible ducks at 1 to 2 days old are immunized by subcutaneous or intramuscular injection of 0.5 ml of the triple-valent and tetravalent inactivated vaccine into the neck.

[0019] Preferably, after immunization, 10 / 10 of the duck hepatitis A virus type 1 test ducks are protected, 9 / 10 or more of the duck hepatitis A virus type 3 test ducks are protected, and the serum neutralizing antibody titer is not less than 1:40; 10 / 10 of the novel reovirus test ducks are protected; 9 / 10 or more of the novel duck parvovirus test ducks are protected.

[0020] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0021] 1. Among the currently commercially available waterfowl vaccines, there is only a bivalent inactivated vaccine for duck hepatitis, and there is no live or inactivated vaccine against novel duck reovirus and novel duck parvovirus. The triple-valent and tetravalent inactivated vaccine prepared by the present invention can not only achieve the purpose of preventing multiple diseases with one injection, reducing the stress reaction of ducks due to immunization with vaccines, but also these four viral diseases are all susceptible to young ducks, and can prevent the invasion of four viruses simultaneously after immunization.

[0022] 2. When the duck hepatitis virus is stored at -80°C, the virus content decreases rapidly and it is difficult to store for a long time. Therefore, when conducting potency tests on the finished product, it is necessary to conduct virulence tests on the virus used for challenge each time in advance. If it does not meet the standard, it needs to be re-prepared, which greatly increases the difficulty and duration of the test. We store the virus used for inspection in liquid nitrogen, and currently, the virus content and virulence have not changed significantly after 36 months of storage (the longer storage period is still in progress).

[0023] 3. Currently, the reported propagation of novel reovirus is all by propagating with adherent cells, harvesting cell cultures, or propagating with embryos, harvesting embryos and allantoic fluid, and grinding to prepare tissue virus, and the titer is generally 10 5.0 ~10 6.0 TCID 50 / 0.1ml (ELD 50 / 0.1 ml). In the present invention, LMH cells were domesticated to obtain a strain of LMH-C cells that can be cultured in suspension, and a propagation process of NDRV on suspended LMH-C cells was successfully explored, increasing the virus content to 10 8.5 TCID 50 / 0.1 ml. Suspended cells do not need to rely on serum for culture, saving production costs and production space, and improving work efficiency. The current virus content is 300 to 3000 times that of cell-derived or embryo-derived virus. The virus cultured without serum has no exogenous proteins, which can reduce the stress response of immunized animals.

[0024] 4. Traditional duck parvovirus can only be propagated in duck embryos or duck embryo fibroblasts, with low yields and high costs. Most research institutions in the literature also mainly focus on the research of inactivated vaccines of the whole virus. The main antigenic epitope of this virus is the VP2 gene. According to the characteristics of this virus, in the present invention, a recombinant baculovirus rSBDS-VP2 strain expressing the VP2 protein of the novel duck parvovirus (commonly known as "duck short dwarf syndrome virus") was constructed using a baculovirus as a vector. The process is as follows: Amplify the VP2 gene with a full length of 1770 bp (optimized), digest and ligate it into the pFastbacHTA vector to obtain a recombinant plasmid containing the VP2 gene. Transform the recombinant plasmid into DH10Bbac competent cells, pick bacmids and perform PCR identification. Transfect the correctly identified bacmids into Sf9 cells. Wait for the cells to swell, increase in volume, and detach, then collect the supernatant and label it as the P1 generation recombinant baculovirus, finally named Re-Bac VP2 strain. Inoculate it into Hi5 insect cells for culture, centrifuge to collect and resuspend the precipitate, ultrasonically disrupt it, and then centrifuge again. Collect the supernatant and use Ni 2+ for chromatography purification to obtain a purified VP2 protein solution with high protein content, a purity of over 80%, good immunogenicity, safety, stability, low production cost, and no biosafety risk in the production process.

[0025] 5. Vaccines prepared according to the aqueous phase formulation ratio and water-oil phase emulsification ratio set in the present invention were used to immunize 10 healthy and susceptible ducks at 3 to 5 days old. Each duck was subcutaneously or intramuscularly injected with 1.0 ml of the vaccine in the neck and observed for 14 days. No local or systemic adverse reactions caused by the vaccine occurred, and the safety was good.

[0026] 6. The vaccine prepared according to the aqueous phase preparation ratio and the water-oil phase emulsification ratio set by the present invention can achieve the best immune protection effect. Among them, 10 / 10 of the test ducks of type 1 duck hepatitis in the immune group are protected, 9 / 10 or more of the test ducks of type 3 duck hepatitis are protected, and the serum neutralizing antibody titer is not less than 1:40. 10 / 10 of the test ducks of novel reovirus are protected, and 9 / 10 or more of the test ducks of novel duck parvovirus are protected (non-excretion). In the control group, 10 / 10 of the test ducks of type 1 duck hepatitis died, 9 / 10 of the test ducks of type 3 duck hepatitis died, 9 / 10 of the test ducks of novel reovirus got sick, and 9 / 10 of the test ducks of novel parvovirus excreted the virus.

[0027] 7. For the currently commercially available bivalent inactivated duck hepatitis vaccine, the serum neutralizing antibody titer in the immune group is not less than 1:24. After immune challenge, 8 / 10 of the test ducks of type 1 and type 3 in the immune group are protected, and 9 / 10 or more of the ducks in the challenge group died, which is lower than the technical effect of the present invention. Detailed implementation mode

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1 Virulent strains for production, engineering cell lines and virulent strains for inspection

[0030] 1.1 Sources The virulent strains of type 1 duck hepatitis virus YBH1 strain, type 3 YBH3 strain, duck reovirus YBAH-03 strain, recombinant baculovirus Re-Bac VP2 strain expressing duck parvovirus VP2 protein (hereinafter referred to as "recombinant baculovirus Re-Bac VP2 strain"), and duck parvovirus YBFJ-02 strain for production and inspection are all isolated / constructed, identified, stored and supplied by Qingdao Yibang Bioengineering Co., Ltd.

[0031] 1.1.1 Isolation and identification of the virulent strain of type 1 duck hepatitis virus YBH1 strain for production and inspection

[0032] 1.1.1.1 Isolation of Duck Hepatitis Virus Type 1 YBH1 Strain for Production and Inspection In around 2009, in a duck farm in Henan, around 10-day-old ducks showed listlessness, convulsions, and acute death. Most of the dead ducks showed opisthotonus and other symptoms. After necropsy of the dead ducks, it was found that the liver was enlarged, brittle and easy to break, mostly yellowish-red, and there were bleeding points or bleeding patches of different sizes on the liver surface. It was preliminarily determined to be duck viral hepatitis. The livers of the diseased ducks were collected, ground, centrifuged, filtered to remove bacteria, and inoculated into the allantoic cavity of 10 - 11-day-old SPF chicken embryos for virus isolation. As a result, after inoculating the chicken embryos, 4 / 5 of the chicken embryos died within 168h; the allantoic fluid and embryos of the dead chicken embryos were collected, homogenized, frozen and thawed 3 times, centrifuged at 4000r / min for 10 minutes, and the supernatant was taken and inoculated into chicken embryos according to the above method. 5 / 5 of the chicken embryos died within 168h; the allantoic fluid and embryos of the dead chicken embryos were collected, homogenized, frozen and thawed 3 times, centrifuged at 4000r / min for 10 minutes, and the supernatant was the harvested virus solution. Samples were taken for PCR, virus content, virulence to chicken embryos, median lethal dose to healthy susceptible ducks, specificity, immune effect, and purity inspection.

[0033] 1.1.1.2 Identification and Results of the Isolated Virus

[0034] 1.1.1.2.1 PCR Identification and Results Extract the virus nucleic acid with reference to the virus DNA / RNA extraction kit instructions. Use the primer sequences designed by our company:

[0035] DHAV-1F: 5'-TCTGATGATTCCTGCTCA-3';

[0036] DHAV-1R: 5'-ATCCATTATATCATCATCAGTTGTG-3';

[0037] DHAV-3F: 5'-CTGTTGAATACGATTGCAACCTGA-3';

[0038] DHAV-3R: 5'-CACTGTCTTCATACAACTATCACCT-3';

[0039] The target band of type 1 is about 750bp long, and the target band of type 3 is about 480bp long. The detection system is 25μL. The reaction program of one-step RT-PCR detection: 50℃ / 30min, 94℃ / 2min, 35×(94℃ / 30s, 56℃ / 30s, 72℃ / 1min), extension at 72℃ for 10min, and finally cooling at 4℃ for 10min. As a result, the target band appeared at 750bp for the isolated virus, and no target band appeared at 480bp.

[0040] 1.1.1.2.2 Virus content determination and results: The isolated virus was serially diluted 10-fold with sterile PBS (0.01 mol / L, pH 7.2 - 7.4, the same below), and 10 -5 、10 -6 、10 -7 、10 -8 dilutions were taken. Five 10 - 11-day-old SPF chicken embryos were inoculated into the allantoic cavity at each dilution, 0.2 ml per embryo; at the same time, five SPF chicken embryos inoculated with sterile PBS as a control were set, 0.2 ml per embryo. Incubation was continued at 36 - 37°C, and the embryos were candled twice a day and observed until 168 hours. The number of dead chicken embryos was counted, and the ELD 50 was calculated by the Reed-Muench method. The result showed that the virus content was 10 7.38 ELD 50 / 0.2 ml.

[0041] 1.1.1.2.3 Virulence to chicken embryos and results: The isolated virus was appropriately diluted with sterile PBS (about 200 ELD 50 / 0.2 ml), and ten 10 - 11-day-old SPF chicken embryos were inoculated into the allantoic cavity, 0.2 ml per embryo. Incubation was continued at 36 - 37°C and observed until 168 hours. Ten chicken embryos died within 48 - 168 hours after inoculation, and all the dead chicken embryos showed thickening and edema of the chorioallantoic membrane, and hemorrhagic lesions on the head, neck, and back.

[0042] 1.1.1.2.4 Determination of the median lethal dose for healthy susceptible ducks and results: The isolated virus was serially diluted 10-fold with sterile PBS, and 10 -4 、10 -5 、10 -6 、10 -7 dilutions were taken. Five 16 - 18-day-old healthy susceptible ducklings were inoculated intramuscularly at each dilution, 0.5 ml per duckling. At the same time, five ducklings inoculated with sterile PBS as a control were set, 0.5 ml per duckling, and observed continuously for 168 hours. The number of dead ducklings was counted, and the median lethal dose (LD 50 ) of ducklings was calculated by the Reed-Muench method. The result showed that the median lethal dose of the isolated virus per 0.5 ml for ducklings was 10 6.0 LD 50 .

[0043] 1.1.1.2.5 Specificity test and results: The isolated virus was appropriately diluted with sterile PBS (about 200 ELD 50(0.2 ml), mixed with an equal volume of anti-Duck Hepatitis Virus Type 1 positive serum (neutralizing antibody titer should not be less than 1:1000), incubated at 37°C for 1 hour for neutralization, then inoculated into 10 SPF chicken embryos at 10 - 11 days old via the allantoic cavity, 0.2 ml per embryo. Meanwhile, 10 virus control embryos were set up, 0.1 ml per embryo (containing approximately 100 ELD 50 ), incubated at 36 - 37°C continuously, and observed until 168 hours. Results: Within 48 - 168 hours, no specific deaths caused by Duck Hepatitis Virus Type 1 occurred in the neutralization group chicken embryos, and 10 / 10 survived; 10 / 10 of the control group chicken embryos died, and the dead chicken embryos all showed thickening and edema of the chorioallantoic membrane, and hemorrhagic lesions on the head, neck, and back.

[0044] 1.1.1.2.6 Immune effect test and results: The isolated virus was diluted 10 - fold (virus content was 10 6.38 ELD 50 / 0.2 ml), inactivated at 37°C for 24 h with formaldehyde solution at a final concentration of 0.2%, and then made into inactivated vaccine according to the ratio of 1

[0045] ︰2 (aqueous phase︰oil phase). Twenty 1 - 2 - day - old healthy and susceptible ducks were used, 10 ducks were each subcutaneously or intramuscularly injected with 0.5 ml of the vaccine, and the other 10 ducks were not immunized as the control. 15 - 16 days after inoculation, 0.5 ml of the isolated virus strain YBH1 (containing 10 4.5 LD 50 ) was intramuscularly injected respectively, and observed for 168 hours. Results: 10 / 10 of the control group died, and 10 / 10 of the immunized group were protected.

[0046] 1.1.1.2.7 Purity test and results: The test was carried out according to the appendix of Volume III of the Chinese Veterinary Pharmacopoeia (2005 edition), the method for checking duck embryo fibroblasts, and the method for checking Duck Hepatitis B Virus. Results showed that the isolated virus had no bacterial growth, no mycoplasma and exogenous virus contamination.

[0047] Finally, it was determined that the isolated virus was Duck Hepatitis Virus Type 1, and this virus was named strain YBH1. According to the method during virus isolation above, this virus was passaged 10 times on SPF chicken embryos and stored as the seed lot.

[0048] 1.1.2 Isolation and identification of Duck Hepatitis Virus Type 3 strain YBH3 for production and testing

[0049] 1.1.2.1 Isolation of Duck Hepatitis Virus Type 3 YBH3 Strain for Production and Inspection In 2013, from a duck flock in Shandong that still showed typical symptoms of duck viral hepatitis after being immunized with Duck Hepatitis Vaccine Type 1, the livers of diseased ducks were collected. After grinding, centrifugation, and filtration to remove bacteria, the allantoic cavities of 12 - 13-day-old duck embryos were inoculated. Dead embryos within 24 hours were discarded, and duck embryos that died earlier after 24 hours were collected. The livers were homogenized, frozen and thawed 3 times, centrifuged at 4000 r / min for 10 minutes, and the supernatant was inoculated into 10 - 11-day-old SPF chicken embryos. The livers of dead chicken embryos were processed as described above and then inoculated into duck embryos. After repeating this process 3 times, a chicken embryo-adapted virus was obtained. This virus was inoculated into the allantoic cavities of 10 - 11-day-old SPF embryos. 4 / 5 of the chicken embryos died within 168 hours. The allantoic fluid and embryos of the dead chicken embryos were collected, homogenized, frozen and thawed 3 times, centrifuged at 4000 r / min for 10 minutes, and the supernatant was used as the virus seed of the isolated virus. Samples were taken for PCR, virus content, virulence to chicken embryos, median lethal dose to healthy susceptible ducks, specificity, immune effect, and purity inspection.

[0050] 1.1.2.2 Identification of the Isolated Virus and Results

[0051] 1.1.2.2.1 PCR Identification and Results PCR detection was carried out according to the method in item 1.1.1.2.1. As a result, the isolated virus showed a target band at 480 bp and did not show a target band at 750 bp.

[0052] 1.1.2.2.2 Virus Content Determination and Results The isolated virus was serially diluted 10-fold with sterile PBS. Dilutions of 10 -5 、10 -6 、10 -7 、10 -8 were taken, and 5 10 - 11-day-old SPF chicken embryos were inoculated into the allantoic cavity at each dilution, 0.2 ml per embryo. At the same time, 5 SPF chicken embryos inoculated with sterile PBS as a control were set, 0.2 ml per embryo. Incubation was continued at 36 - 37°C, and the embryos were candled twice a day and observed until 168 hours. The number of dead chicken embryos was counted, and the ELD 50 was calculated by the Reed-Muench method. As a result, the virus content was 10 7.0 ELD 50 / 0.2 ml.

[0053] 1.1.2.2.3 Virulence Determination to Chicken Embryos and Results The isolated virus was appropriately diluted with sterile PBS (about containing 200 ELD 50 / 0.2 ml), and 10 10 - 11-day-old SPF chicken embryos were inoculated into the allantoic cavity, 0.2 ml per embryo. Incubation was continued at 36 - 37°C and observed until 168 hours. 8 of the chicken embryos died within 48 - 168 hours after inoculation, and the dead chicken embryos all showed thickening and edema of the chorioallantoic membrane, and hemorrhagic lesions on the head, neck, and back.

[0054] 1.1.1.2.4 Determination of the median lethal dose for healthy susceptible ducks and results The isolated virus was serially diluted 10-fold with sterile PBS. Take 10 -4 、10 -5 、10 -6 、10 -7 4 dilutions. For each dilution, 5 healthy susceptible ducklings at 16 - 18 days old were inoculated intramuscularly, 0.5 ml per duckling. At the same time, 5 ducks inoculated with sterile PBS as a control, 0.5 ml per duckling, were continuously observed for 168 hours. The number of dead ducklings was counted, and the median lethal dose (LD 50 ) of ducklings was calculated by the Reed-Muench method. As a result, the median lethal dose of the isolated virus per 0.5 ml for ducklings was 10 5.83 LD 50 .

[0055] 1.1.1.2.5 Specificity test and results The isolated virus was appropriately diluted with sterile PBS (about 200 ELD 50 / 0.2 ml), mixed with an equal volume of positive serum against duck hepatitis virus type 3 (the neutralizing antibody titer should not be less than 1:1000), incubated at 37°C for 1 hour, and then inoculated into 10 SPF chicken embryos at 10 - 11 days old via the allantoic cavity, 0.2 ml per embryo. At the same time, 10 chicken embryos were set as a virus control, 0.1 ml per embryo (about 100 ELD 50 ), and incubated at 36 - 37°C. Observation was continued until 168 hours. As a result, within 48 - 168 hours, no specific death caused by duck hepatitis virus type 3 occurred in the neutralized group of chicken embryos, and 10 / 10 survived; 8 / 10 of the control group of chicken embryos died, and the dead chicken embryos all showed thickening and edema of the chorioallantoic membrane and hemorrhagic lesions on the head, neck, and back.

[0056] 1.1.1.2.6 Immune effect test and results The isolated virus was diluted 10-fold with sterile PBS (the virus content was 10 6.0 ELD 50 / 0.2 ml), inactivated at 37°C for 24 hours with a formaldehyde solution at a final concentration of 0.2%, and then made into an inactivated vaccine at a ratio of 1:2 (aqueous phase: oil phase) after inactivation. Twenty healthy susceptible ducklings at 1 - 2 days old were used. Ten of them were each subcutaneously or intramuscularly injected with 0.5 ml of the vaccine, and the other ten were not immunized as a control. At 15 - 16 days after inoculation, 0.5 ml of the isolated virus strain YBH3 (containing 10 4.5 LD 50 ) was intramuscularly injected respectively, and observed for 168 hours. As a result, 9 / 10 of the control group died, and 10 / 10 of the immunized group were protected.

[0057] 1.1.1.2.7 Purity inspection and results: The inspection was carried out according to the appendix of the third part of the Chinese Veterinary Pharmacopoeia (2010 edition), the duck embryo fibroblast inspection method, and the duck hepatitis B virus inspection method. As a result, no growth of the isolated virus was observed, and there was no contamination by mycoplasma or exogenous viruses.

[0058] Finally, it was determined that the isolated virus was duck hepatitis virus type 3, and this isolated virus was named strain YBH 3. According to the above passage method, the virus was passaged 10 times on SPF chicken embryos and stored as a seed lot.

[0059] 1.1.3 Isolation and identification of the novel duck reovirus strain YBH-03 for production and inspection

[0060] 1.1.3.1 Isolation of the novel duck reovirus strain YBH-03 for production and inspection: In 2017, after necropsy of dead ducks in a duck farm in Anhui, symptoms such as enlarged spleen, patchy necrosis, irregular necrosis and hemorrhage of the liver, and myocardial hemorrhage were mainly found. The overall mortality rate of the flock was about 10%. Diseased spleens and livers of the diseased ducks were collected, ground, centrifuged, filtered and sterilized, and then inoculated into LMH-C adherent cells. Obvious cytopathic effects appeared after 2 passages. When the cytopathic effects reached more than 80%, the cells were frozen and thawed 2 times, and the cell culture was harvested. Samples were taken for PCR, virus content, virulence to ducklings, specificity, immune effect and purity inspection.

[0061] 1.1.3.2 Identification of the isolated virus and results

[0062] 1.1.3.2.1 PCR identification and results: The viral nucleic acid was extracted with reference to the instructions of the virus DNA / RNA extraction kit, and the primer sequences designed by our company were used:

[0063] NDRV-F: 5'-CGATGAATGGTGGAACGCCTG-3';

[0064] NDRV-R: 5'-AGCGTACGACCCTACGCCT-3';

[0065] The target band was about 316 bp in length. The detection system was 25 μL. The reaction program for one-step RT-PCR detection was: 50 °C / 30 min, 94 °C / 2 min, 35×(94 °C / 30 s, 56 °C / 30 s, 72 °C / 1 min), 72 °C extension for 5 min, and finally cooling at 4 °C for 10 min. As a result, the target band appeared at 316 bp for the isolated virus.

[0066] 1.1.3.2.2 Determination of virus content and results: The isolated virus was serially diluted 10-fold with cell maintenance medium (DMEM / F12 containing 2% fetal bovine serum); take 10 -5 、10 -6 、10 -7 、10-8 Four dilutions were respectively inoculated onto a 96-well plate of well-grown adherent LMH-C cells. Six wells were inoculated for each dilution, 0.1 ml per well, and they were placed in an incubator at 37°C containing 5% CO 2 and continuously cultured for 7 days. The cytopathic effect was observed and recorded, and the TCID 50 . As a result, the virus content was 10 6.0 TCID 50 / 0.1 ml.

[0067] 1.1.3.2.3 Virulence determination for ducklings and results The isolated virus was diluted with sterile PBS to 10 5.0 TCID 50 / 0.1 ml, and 10 healthy and susceptible ducks at 16 - 18 days old were inoculated intramuscularly, 0.1 ml per duck. They were sacrificed on the 4th day after inoculation, and whether the spleen was enlarged or necrotic was observed. After processing the spleen, virus isolation was performed using adherent LMH-C cells. As a result, 10 / 10 of the spleens of the experimental ducks had obvious enlargement or necrosis; virus isolation was performed on the spleens of the experimental ducks, and 9 / 10 were positive for virus isolation.

[0068] 1.1.3.2.4 Specificity test and results The isolated virus was diluted with serum-free DMEM / F12 medium to 100 TCID 50 / 0.1 ml, mixed with an equal volume of anti-duck reovirus positive serum (neutralizing antibody titer not less than 1:10), neutralized at 37°C for 1 hour, inoculated onto a 96-well cell culture plate of well-grown adherent LMH-C cells, 10 wells were inoculated, 0.2 ml per well, cultured at 37°C for 1 - 2 hours, the virus solution was discarded, washed 3 times with serum-free DMEM / F12 medium, and 0.2 ml of cell maintenance medium was added to each well; at the same time, virus control and maintenance medium control were set, 10 wells were inoculated for each, 0.1 ml per well. They were placed in an incubator at 37°C containing 5% CO 2 and cultured for 7 days, and the cytopathic effect (CPE) was observed and recorded. As a result, CPE occurred in all virus control wells, and no CPE appeared in the neutralized wells and maintenance medium control wells.

[0069] 1.1.3.2.5 Immunization effect test and results The isolated virus was diluted 316-fold with sterile PBS (virus content was 10 3.5 TCID 50 / 0.1 ml), inactivated with formaldehyde solution at a final concentration of 0.2% at 37°C for 24 h, and an inactivated vaccine was prepared according to the ratio of 1:2 (aqueous phase: oil phase) after inactivation. Twenty healthy and susceptible breeding ducks at 1 - 2 days old were used, 10 ducks were each subcutaneously or intramuscularly injected with 0.5 ml of the vaccine, and the other 10 ducks were not immunized as a control. At 15 - 16 days after immunization, they were respectively intramuscularly injected with the virus solution of duck reovirus strain YBAH-03 for challenge, 0.1 ml per duck (containing 105.0 TCID 50 ) and they were sacrificed on the 4th day after inoculation. Results: In the control ducks, 10 / 10 spleens showed varying degrees of swelling or necrosis; in the immunized ducks, 10 / 10 spleens were not swollen and not necrotic. Spleens of the experimental ducks were collected for virus isolation. Virus isolation was positive in 9 / 10 of the control ducks; virus isolation was negative in 10 / 10 of the immunized ducks.

[0070] 1.1.3.2.6 Purity test and results The test was carried out according to the appendix of the third part of the Chinese Veterinary Pharmacopoeia (2015 edition), the duck embryo fibroblast examination method, and the duck hepatitis B virus examination method. Results: The isolated virus showed no growth in sterile conditions, no mycoplasma contamination, and no exogenous virus contamination.

[0071] Based on the results of virus isolation and identification, it was confirmed that the strain was a novel duck reovirus strain, named YBAH-03 strain. According to the above passage method, the virus was passaged 10 times on LMH-C adherent cells and stored as a seed lot.

[0072] 1.1.4 Construction and identification of the recombinant baculovirus Re-Bac VP2 strain expressing the VP2 protein of novel duck parvovirus

[0073] 1.1.4.1 Optimization and synthesis of the VP2 gene The VP2 gene of the novel duck parvovirus isolate YBFJ-02 strain was optimized for codons, GC content, etc., and the optimized gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0074] 1.1.4.2 Enzyme digestion reaction

[0075] 1.1.4.2.1 Samples were added to a 1.5 ml centrifuge tube according to the following reaction system. The reaction system was 50 μL:

[0076] Name Volume (μl) pFastbacHTA vector / plasmid containing target gene VP2 10 (2 μg) EcoRⅠ 2 HindⅢ 2 10×Buffer 5 <![CDATA[ddH 2 O]]> Make up to 50

[0077] 1.1.4.2.2 The 1.5 ml centrifuge tube in step 1.1.4.2.1 was placed in a 37 °C constant temperature water bath for 15 min.

[0078] 1.1.4.2.3 Gel recovery of the double enzyme digestion products The above double enzyme digestion system was taken out, and agarose gel electrophoresis was carried out according to the instructions of the OMEGA gel recovery kit to recover the DNA fragment.

[0079] 1.1.4.3 Ligation reaction Samples were added to a 0.2 ml centrifuge tube according to the following reaction system:

[0080]

[0081]

[0082] After sample addition is completed, gently pipette up and down several times to mix all components. Place the 0.2 ml centrifuge tube at 4 °C for overnight reaction. After the reaction is completed, directly proceed with the transformation experiment.

[0083] 1.1.4.4 Transformation reaction According to the instruction manual of Takara JM109 competent cells, transform the ligation product into competent cells.

[0084] 1.1.4.5 Plasmid extraction

[0085] (1) Use a 10 μL pipette tip to pick a single colony from the transformation plate into 3 ml of LB liquid medium containing ampicillin resistance, and shake the bacteria overnight at 37 °C and 220 rpm.

[0086] (2) Aspirate the bacterial solution into a 1.5 ml centrifuge tube, centrifuge at room temperature at 5000 rpm for 3 min, and discard the supernatant.

[0087] (3) According to the instruction manual of the OMEGA plasmid extraction kit, extract the plasmid and dissolve it in 30 μL of Elution buffer. The extracted plasmid can be used for the next PCR identification.

[0088] 1.1.4.6 PCR identification

[0089] 1.1.4.6.1 Add samples and mix according to the following table. The reaction system is 25 μL:

[0090] F: 5'-GAATTCATGACGGCTCCTGCAAAAAA-3';

[0091] R: 5'-AAGCTTTTACAGATTTTGAGTTAGAT-3'; The target band is about 210 bp long.

[0092] Name Volume (μl) Forward primer (10 uM) 0.5 Reverse primer (10 uM) 0.5 2×Mix 12.5 Plasmid template 0.5 <![CDATA[ddH 2 O]]> Make up to 25

[0093] 1.1.4.6.2 PCR amplification program: 98 °C: 2 min; 98 °C: 10 s, 55 °C: 30 s, 72 °C: 1 min (31 cycles) 72 °C: 5 min.

[0094] 1.1.4.6.3 Sequencing: Send the plasmid with positive PCR identification to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0095] 1.1.4.7 Transformation According to the instruction manual of Tsingke DH10bac competent cells, transform the plasmid with positive sequencing into DH10bac competent cells.

[0096] 1.1.4.8 Colony Picking Using a 10 μL pipette tip, pick white monoclonal colonies from the transformed plate in 1.1.4.7 and transfer them to 5 mL of LB liquid medium containing kanamycin resistance, tetracycline resistance, and gentamicin resistance. Incubate the culture at 37 °C with shaking at 220 rpm overnight. Extract the plasmid according to the instruction manual of the bacmid small-scale extraction kit for baculovirus shuttle vectors.

[0097] 1.1.4.9 PCR Identification The method is the same as that in 1.1.4.6.

[0098] 1.1.4.10 Packaging of Baculovirus

[0099] (1) Preparation: Preheat the TNM-FH culture medium in a 27 °C water bath to 27 °C.

[0100] (2) Add 2 μg of recombinant DNA to 100 μL of TNM-FH culture medium without serum and antibiotics, and mix well. Add 9 μL of Cellfectin Reagent to 100 μL of TNM-FH culture medium without serum and antibiotics, and mix well. Mix the liposome with the recombinant DNA and let it stand at room temperature for 40 min.

[0101] (3) Take out the 6-well plate cells from the 27 °C incubator, discard the supernatant medium, wash the cells three times with pre-warmed TNM-FH culture medium, and discard the TNM-FH culture medium.

[0102] (4) Add 2 mL of TNM-FH culture medium with 10% fetal bovine serum to each cell well.

[0103] (5) Gently add the mixture of recombinant DNA and liposome to each well of the cells, mix gently, and incubate statically at 27 °C for 5 - 6 h.

[0104] (6) Discard the liquid in the wells, add 2 mL of complete TNM-FH culture medium (containing antibiotics and 10% serum), and incubate statically at 27 °C for 5 - 6 days.

[0105] (7) When the cells swell, increase in volume, and detach, collect the supernatant, label it as the P1 generation of recombinant baculovirus, and name it SBDSV-VLP-P.

[0106] (8) Infect newly cultured Sf9 cells with SBDSV-VLP-P1 to increase the content of recombinant baculovirus. After continuous inoculation for 2 generations, collect the cell supernatant and store it at 4 °C or -20 °C for later use.

[0107] 1.1.4.11 Protein Purification and Detection

[0108] 1.1.4.11.1 Expression and Identification of Recombinant Baculovirus VP2 Protein in Insect Cells Sf9 The prepared recombinant baculovirus was used to infect insect cells Sf9, and the cells were cultured at 27 °C for 72 h. At the same time, normal uninfected insect cells Sf9 were cultured at 27 °C for 72 h as a control. The cells were harvested, and the culture supernatant was frozen for later use. After the cells were washed with PBS at pH 7.4, 1× SDS-PAGE loading buffer [50 mM Tris-HCl (pH 6.8), 100 mM Dithiothreitol (DTT), 2% SDS, 0.05% Bromophemol blue, 10% Glycerol] was added, and the mixture was boiled for 5 min. Polyacrylamide gel electrophoresis was performed using a 12% separating gel and a 5% stacking gel at 100 V for about 2.5 h, and then stained with Coomassie Brilliant Blue R250. The results showed that VP2 protein was expressed in the lysate of insect cells Sf9 infected with the recombinant baculovirus.

[0109] 1.1.4.11.2 Chromatographic Purification of VP2 Protein The recombinant baculovirus SBDSV-VLP-P1 was inoculated into insect cells Sf9. After culturing at 27 °C for 72 h, the cell precipitate was collected by centrifugation. An appropriate amount of PBS was added to the cell precipitate to resuspend the cells. The cells were lysed by ultrasonic treatment and centrifuged at 1000 r / min at 4 °C for 10 min. The supernatant was collected and purified by chromatography using a Ni2+ column.

[0110] 1.1.4.11.3 Western Blot Assay The prepared protein was subjected to SDS-PAGE, and transferred by semi-dry transfer at 20 V for 30 min to transfer the target protein band to a PVDF membrane. The transfer membrane was blocked with a blocking solution overnight, washed 3 times with PBST, incubated with a 1:500 diluted positive serum of duck parvovirus at 37 °C for 1.5 h, washed 3 times with PBST, incubated with a 1:2000 diluted HRP-labeled rabbit anti-duck enzyme-labeled antibody at 37 °C for 1.5 h, washed 3 times with PBST, and incubated with a substrate solution for 5 min, and then developed on a chemiDOC. As a result, a specific band was detected at about 65 kDa, indicating that the VP2 protein band expressed by the VP2 gene in insect cells Sf9 was very bright.

[0111] 1.1.4.11.4 Electron Microscopy Observation The expression product purified by chromatography using a Ni 2+ column was negatively stained and observed by electron microscopy to observe the shape and size of the expressed protein. As a result, under the electron microscope, the shape of the protein was hexagonal and the size was about 20 nm.

[0112] 1.1.4.11.5 Virus Titer Determination and Results The virus strain was serially diluted 10-fold with a commercial serum-free medium for insect cells. Take 10 -4 、10 -5 、10 -6, 10 -7 Four dilutions were respectively inoculated into a 96-well culture plate with well-grown monolayer Sf9 insect cells. Six wells were inoculated for each dilution, 0.1 ml per well. Meanwhile, six wells of normal cell control were set up. After culturing at 27 °C for 96 hours, the culture medium was discarded and indirect immunofluorescence assay was performed. The TCID was calculated by the Reed-Muench method 50 . As a result, the virus content was 10 6.50 TCID 50 / 0.1 ml.

[0113] 1.1.4.11.6 Protein content determination and results In the bioreactor, Hi5 insect cells were amplified and cultured to a cell density of 6.0×10 6 ~7.0×10 6 cells / ml, and then the cell density was diluted to 2.5×10 6 cells / ml with serum-free insect cell medium. The recombinant baculovirus Re-Bac VP2 strain used for production was inoculated at an MOI of 0.4. The culture was carried out at 27 °C under the condition that the dissolved oxygen value was 40%, and the stirring speed was adjusted to the appropriate rotation speed matching the bioreactor. After culturing for 72 - 76 hours, the VP2 protein solution was harvested after standing and centrifugation. As a result, the detected VP2 protein content was 517 μg / ml and the purity was 87%.

[0114] 1.1.4.11.7 Immunization effect test and results The protein solution harvested in 1.1.4.11.6 was used, and the content of the target protein was adjusted to 100 μg / ml with sterilized PBS solution. It was inactivated with a formaldehyde solution with a final concentration of 0.1% at 37 °C for 48 h, and then inactivated vaccine was prepared at a ratio of 1:2 (aqueous phase: oil phase). Twenty healthy and susceptible ducks at 1 - 2 days old were used. Ten ducks were subcutaneously or intramuscularly injected with 0.5 ml of the vaccine in the neck, and ten ducks were not immunized as a control. 15 - 16 days after immunization, 0.2 ml of duck parvovirus YBFJ-02 strain

[0115] (containing 10 5.5 ELD 50 ) was intramuscularly injected respectively. Seven days after virus challenge, anal swabs were collected from all experimental ducks and processed for PCR detection. As a result, 9 / 10 of the non-immunized and virus-challenged group were PCR positive, and 10 / 10 of the immunized group were PCR negative.

[0116] 1.1.4.11.8 Purity test and results The test was carried out according to the appendix of the third part of the Chinese Veterinary Pharmacopoeia (2015 edition). As a result, there was no bacterial growth, no mycoplasma and exogenous virus contamination.

[0117] Finally, the recombinant baculovirus expressing the novel duck parvovirus VP2 protein was named Re-Bac VP2 strain, and according to the above culture method, it was continuously passaged 8 generations to establish a seed lot.

[0118] 1.1.5 Isolation and Identification of the Novel Duck Parvovirus Strain YBFJ - 02 for Virulence Testing

[0119] 1.1.5.1 Isolation of the Novel Duck Parvovirus Strain YBFJ - 02 for Virulence TestingIn 2015, a new disease occurred in Cherry Valley ducks in a duck farm in Fujian, with clinical manifestations such as short beak, protruding tongue, soft legs, poor feather growth, easy fracture, and growth retardation. The livers and spleens of the diseased ducks were collected, ground, and subjected to PCR detection, sequencing, and alignment. It was found that its homology with the GPV of the Hungarian strain was over 80%, and it was preliminarily determined to be a parvovirus. The ground diseased material was frozen and thawed 3 times, centrifuged at 8000 r / min for 10 minutes, and the supernatant was filtered through a 0.22 μm filter and then inoculated into the yolk sac of healthy and susceptible 9 - 10 - day - old duck embryos. The embryos were incubated at 36 - 37 °C, candled twice a day, and observed until 216 hours. The allantoic fluid of the dead embryos was collected and inoculated into susceptible duck embryos according to the above method. After 3 passages, the death of duck embryos was relatively stable, and the allantoic fluid of the dead embryos was harvested. Samples were taken for PCR, virus content, virulence to duck embryos, virulence to healthy and susceptible ducks, specificity, and purity tests.

[0120] 1.1.3.2 Identification of the Isolated Virus and Results

[0121] 1.1.3.2.1 PCR Identification and ResultsViral nucleic acid was extracted with reference to the instructions of the virus DNA / RNA extraction kit. Using the primer sequences designed by our company:

[0122] SBDSV - F: 5'-CAATGGGCTTTTACCAATATGC-3';

[0123] SBDSV - R: 5'-ATTTTTCCCTCCTCCCACCA-3';

[0124] The target band was about 640 bp long. The detection system was 25 μL. The reaction program for one - step PCR detection was: 95 °C / 3 min, 35×(98 °C / 10 s, 55 °C / 30 s, 72 °C / 1 min), 72 °C extension for 5 min, and finally cooled to 4 °C for 10 min. As a result, the target band appeared at 640 bp for the isolated virus.

[0125] 1.1.3.2.2 Determination of Virus Content and ResultsThe isolated virus was serially diluted 10 - fold with sterile PBS, and 10 -5 、10 -6 、10 -7 、10 -8Four dilutions were made. Five susceptible duck embryos at 9 - 10 days of age were inoculated with 0.2 ml of each dilution on the chorioallantoic membrane. At the same time, five susceptible duck embryos inoculated with sterilized PBS (0.2 ml per embryo) were set as the control. Incubation was continued at 36 - 37 °C, and the embryos were candled twice a day and observed for 216 hours. The number of dead duck embryos was counted, and the ELD was calculated by the Reed - Muench method. 50 As a result, the virus content was 10 7.0 ELD 50 / 0.2 ml.

[0126] 1.1.3.2.3 Virulence determination of the isolated virus on duck embryos and results The isolated virus was appropriately diluted with sterilized PBS (about 200 ELD 50 / 0.2 ml). Ten susceptible duck embryos at 9 - 10 days of age were inoculated with 0.2 ml of the diluted virus on the chorioallantoic membrane. Incubation was continued at 36 - 37 °C and observed for 216 hours. As a result, 9 duck embryos died within 24 - 216 hours after inoculation, and all dead duck embryos showed lesions of thickening and edema of the chorioallantoic membrane.

[0127] 1.1.3.2.4 Virulence determination of the isolated virus on healthy susceptible ducklings and results The isolated virus was diluted 30 - fold with sterilized PBS and 10 healthy susceptible ducklings at 16 - 18 days of age were intramuscularly injected with 0.2 ml each (containing 10 5.5 ELD 50 ). At the same time, 5 ducklings of the same batch were set as the non - inoculated control. Observation was continued for 7 days. Anal swabs of all test ducklings were collected on the 7th day and processed for PCR detection. As a result, all PCR detections of the control ducklings were negative, and all PCR detections of the challenged ducklings were positive.

[0128] 1.1.3.2.5 Specificity test and results The virus strain was appropriately diluted with sterilized PBS (about 200 ELD 50 / 0.2 ml), mixed with an equal volume of anti - duck parvovirus positive serum, incubated at 37 °C for 1 hour, and then 10 susceptible duck embryos at 9 - 10 days of age were inoculated with 0.2 ml of the mixture on the chorioallantoic membrane. At the same time, 10 virus controls (0.1 ml per embryo, about 100 ELD 50 ) were set. Incubation was continued at 36 - 37 °C and observed for 216 hours. As a result, within 24 - 216 hours, 10 duck embryos in the neutralization group survived; 9 duck embryos in the control group died, and all dead duck embryos showed lesions of thickening and edema of the chorioallantoic membrane.

[0129] 1.1.3.2.6 Purity test and results The test was carried out according to the appendix of the third part of the Chinese Veterinary Pharmacopoeia (2015 edition), the method for examining duck embryo fibroblasts, and the method for examining duck hepatitis B virus. As a result, no bacteria grew in the isolated virus, and there was no contamination with mycoplasma and exogenous viruses.

[0130] According to the results of virus isolation and identification, it was confirmed that the strain was a novel duck parvovirus, named strain YBFJ-02. According to the above passage method, the virus was passaged 10 times continuously on Jiangkang susceptible duck embryos and stored as the seed lot.

[0131] It should be noted that the above-mentioned type 1 duck hepatitis virus strain is strain YBH 1, which was deposited in the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: V202513;

[0132] The type 3 duck hepatitis virus strain is strain YBH 3, which was deposited in the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: V202514;

[0133] The duck reovirus strain is strain YBAH-03, which was deposited in the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: V202511;

[0134] The recombinant baculovirus Re-Bac VP2 strain expressing duck parvovirus VP2 protein was deposited in the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCCNO: V202512;

[0135] The chicken liver cancer suspension cell LMH-C was deposited in the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: C202558.

[0136] After each virus seed was stored for 36 to 60 months, it was tested according to the above identification method, and the standards for each virus seed were formulated as follows:

[0137] Standard of strain 2

[0138] 2.1 Type 1 duck hepatitis virus YBH1 strain

[0139] 2.1.1 Virus content The virus strain was serially diluted 10-fold with sterilized PBS (0.01 mol / L, pH 7.2 - 7.4, the same below), and 10 -5 、10 -6 、10 -7 、10 -8 4 dilution factors were taken, and 5 10 - 11-day-old SPF chicken embryos were inoculated into the allantoic cavity at each dilution factor, 0.2 ml per embryo; at the same time, 5 SPF chicken embryos inoculated with sterilized PBS were set as controls, 0.2 ml per embryo. Incubate at 36 - 37 °C and continue to incubate, candling the embryos twice a day, and observing until 168 hours. Count the number of dead chicken embryos (the dead chicken embryos should show thickening and edema of the chorioallantoic membrane, and hemorrhagic lesions on the head, neck, and back), and calculate the ELD50 The virus content per 0.2 ml should be not less than 10 6.0 ELD 50 .

[0140] 2.1.2 Virulence to chicken embryos Dilute the virus strain appropriately with sterile PBS (about 200 ELD 50 / 0.2 ml), inoculate 10 10- to 11-day-old SPF chicken embryos via the allantoic cavity at 0.2 ml per embryo, incubate at 36 - 37 °C, and observe for 168 hours. At least 8 of the chicken embryos should die within 48 - 168 hours after inoculation, and the dead chicken embryos should show thickening and edema of the chorioallantoic membrane, and hemorrhagic lesions on the head, neck, and back.

[0141] 2.1.3 Median lethal dose to healthy susceptible ducks Dilute the virus strain serially 10-fold with sterile PBS, take 10 -3 , 10 -4 , 10 -5 , 10 -6 4 dilution factors, intramuscularly inoculate 5 16- to 18-day-old healthy susceptible ducklings at each dilution factor at 0.5 ml per duckling. At the same time, set up a control group of 5 ducklings inoculated with sterile PBS at 0.5 ml per duckling, and observe continuously for 168 hours. Count the number of dead ducklings and calculate the median lethal dose (LD 50 ). Each 0.5 ml should be not less than 10 4.5 LD 50 .

[0142] 2.1.4 Pathogenicity to healthy susceptible ducklings Dilute the virus strain 10-fold with sterile PBS and intramuscularly inoculate 10 16- to 18-day-old healthy susceptible ducklings at 0.5 ml per duckling. At least 8 of the ducklings should die within 168 hours.

[0143] 2.1.5 Specificity Dilute the virus strain appropriately with sterile PBS (about 200 ELD 50 / 0.2 ml), mix it with an equal volume of positive serum against duck hepatitis virus type 1 (the neutralizing antibody titer should be not less than 1:1000), incubate at 37 °C for 1 hour for neutralization, then inoculate 10 10- to 11-day-old SPF chicken embryos via the allantoic cavity at 0.2 ml per embryo. At the same time, set up a virus control group of 10 embryos inoculated at 0.1 ml per embryo (about 100 ELD 50 ), incubate at 36 - 37 °C, and observe for 168 hours. Within 48 - 168 hours, the chicken embryos in the neutralization group should not show specific death caused by duck hepatitis virus type 1 and at least 8 should survive; the chicken embryos in the control group should have at least 8 deaths, and the dead chicken embryos should show thickening and edema of the chorioallantoic membrane, and hemorrhagic lesions on the head, neck, and back.

[0144] 2.1.6 Immunogenicity Adjust the virus content to 10 6.0 ELD 50 / 0.2 ml of the virus solution of Duck Hepatitis Virus Type 1 strain YBH1, after inactivated, is made into an inactivated vaccine according to the ratio of 1:2 (aqueous phase: oil phase). Twenty healthy and susceptible ducks at 1 - 2 days old are used. Ten of them are subcutaneously or intramuscularly injected with 0.5 ml of the vaccine each, and the other ten are not immunized as a control. 15 - 16 days after inoculation, 0.5 ml of the challenge virus of Duck Hepatitis Virus Type 1 strain YBH1 (containing 10 4.5 LD 50 ) is intramuscularly injected respectively, and observed for 168 hours. At least 8 ducks in the control group should die, and at least 8 ducks in the immunized group should be protected.

[0145] 2.1.7 Purity It is examined according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia, the examination method of duck embryo fibroblast cells and the examination method of duck hepatitis B virus, and there should be no growth of bacteria, no contamination of mycoplasma and exogenous virus.

[0146] 2.1.8 Basic seed passage The basic seed passage for production is from E7 to E9 generations; the basic seed passage for challenge is from E5 to E7 generations.

[0147] 2.1.9 Strain preservation It is preserved in liquid nitrogen.

[0148] 2.2 Duck Hepatitis Virus Type 3 strain YBH3

[0149] 2.2.1 Virus content The strain is serially diluted 10 - fold with sterilized PBS. Take 10 -5 、10 -6 、10 -7 、10 -8 4 dilution degrees. Five 10 - 11 - day - old SPF chicken embryos are inoculated into the allantoic cavity at each dilution degree, 0.2 ml per embryo; at the same time, five SPF chicken embryos inoculated with sterilized PBS as a control are inoculated, 0.2 ml per embryo. Incubate at 36 - 37 °C continuously, candling the embryos twice a day, and observe for 168 hours. Calculate the ELD 50 according to the number of dead duck embryos (the dead duck embryos should show thickening and edema of the chorioallantoic membrane, and hemorrhagic lesions on the head, neck and back). The virus content per 0.2 ml should not be less than 10 6.0 ELD 50 .

[0150] 2.2.2 Virulence to chicken embryos The strain is appropriately diluted with sterilized PBS (about containing 200 ELD 50 / 0.2 ml). Ten 10 - 11 - day - old SPF chicken embryos are inoculated into the allantoic cavity, 0.2 ml per embryo, and incubated at 36 - 37 °C continuously, and observed for 168 hours. At least 8 of the chicken embryos should die within 48 - 168 hours after inoculation, and the dead chicken embryos should show thickening and edema of the chorioallantoic membrane, and hemorrhagic lesions on the head, neck and back.

[0151] 2.2.3 Median lethal dose for healthy susceptible ducks Dilute the virus strain serially 10-fold with sterile PBS. Take 10 -3 、10 -4 、10 -5 、10 -6 4 dilution factors. For each dilution factor, intramuscularly inoculate 5 healthy susceptible ducklings aged 16 - 18 days, 0.5 ml per duckling. At the same time, set up a control group of 5 ducklings inoculated with sterile PBS, 0.5 ml per duckling. Continuously observe for 168 hours. Count the number of dead ducklings and calculate the median lethal dose (LD 50 ). Each 0.5 ml should not be less than 10 4.5 LD 50 .

[0152] 2.2.4 Pathogenicity for healthy susceptible ducklings Dilute the virus strain 10-fold with sterile PBS and intramuscularly inoculate 10 SPF chicken embryos aged 16 - 18 days, 0.5 ml per embryo. At least 8 ducklings should die within 168 hours.

[0153] 2.2.5 Specificity Dilute the virus strain appropriately with sterile PBS (about 200 ELD 50 / 0.2 ml), mix it with an equal volume of positive serum against duck hepatitis virus type 3 (neutralizing antibody titer not less than 1:1000, note 7), incubate at 37°C for 1 hour for neutralization, then inoculate 10 SPF chicken embryos aged 10 - 11 days via the allantoic cavity, 0.2 ml per embryo. At the same time, set up a virus control group of 10 embryos, 0.1 ml per embryo (about 100 ELD 50 ), and continue to incubate at 36 - 37°C and observe until 168 hours. Within 48 - 168 hours, the neutralized group of duck embryos should not show specific death caused by duck hepatitis virus type 3 and at least 8 embryos should survive; the control group of duck embryos should have at least 8 deaths, and the dead duck embryos should show thickening and edema of the chorioallantoic membrane, and hemorrhagic lesions on the head, neck, and back.

[0154] 2.2.6 Immunogenicity Adjust the virus content of the virus solution of duck hepatitis virus type 3 strain YBH3 to 10 6.0 ELD 50 / 0.2 ml, inactivate it and prepare an inactivated vaccine at a ratio of 1:2 (aqueous phase: oil phase). Use 20 healthy susceptible ducklings aged 1 - 2 days, 10 of them are subcutaneously or intramuscularly inoculated with 0.5 ml of the vaccine, and the other 10 are not immunized as a control. 15 - 16 days after inoculation, intramuscularly inoculate 0.5 ml of the challenge virus of duck hepatitis virus type 3 strain YBH3 (containing 10 4.5 LD 50 ) and observe for 168 hours. At least 8 ducklings in the control group should die, and at least 8 ducklings in the immunized group should be protected.

[0155] 2.2.7 Purity It shall be examined according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia, the duck embryo fibroblast inspection method, and the duck hepatitis B virus inspection method, and there shall be no growth of bacteria, no mycoplasma contamination, and no exogenous virus contamination.

[0156] 2.2.8 Basic seed passage The basic seed passage for production is passage E7 - E9; the challenged basic seed passage is passage E5 - E7.

[0157] 2.2.9 Strain preservation It is preserved in liquid nitrogen.

[0158] 2.3 Duck reovirus strain YBAH - 03

[0159] 2.3.1 Virus content The seed virus is serially diluted 10 - fold with cell maintenance medium (DMEM / F12 containing 2% fetal bovine serum); take 10 -4 、10 -5 、10 -6 、10 -7 4 dilution degrees, and inoculate them onto a 96 - well plate of LMH - C cells with good growth, inoculate 5 wells for each dilution degree, 0.1 ml per well, and place it in a cell incubator at 37°C with 5% CO 2 and continue to culture for 7 days, observe and record the cytopathic effect, and calculate the TCID 50 according to the Reed - Muench method. The virus content per 0.1 ml shall not be less than 10 5.0 TCID 50 .

[0160] 2.3.2 Virulence to ducklings The seed virus is diluted with sterile PBS to 10 5.0 TCID 50 / 0.1 ml, and intramuscularly inoculate 10 healthy and susceptible ducklings at 16 - 18 days old, 0.1 ml per duck. Dissect on the 4th day after inoculation, and the spleens of at least 8 ducks should be significantly enlarged or necrotic; collect the spleens of the test ducks for virus isolation, and at least 8 virus isolations should be positive.

[0161] 2.3.3 Specificity The seed virus is diluted with serum - free DMEM / F12 medium to 100 TCID 50 / 0.1 ml, mix it with an equal volume of anti - duck reovirus positive serum (neutralizing antibody titer not less than 1:10), neutralize at 37°C for 1 hour, inoculate a 96 - well cell culture plate of LMH - C cells with good growth, inoculate 10 wells, 0.2 ml per well, culture at 37°C for 1 - 2 hours, discard the virus solution, wash 3 times with serum - free DMEM / F12 medium, and add 0.2 ml of cell maintenance medium to each well; at the same time, set up a virus control and a maintenance medium control, each inoculate 10 wells, 0.1 ml per well. Place it at 37°C with 5% CO 2Cultivate for 7 days in a cell incubator, observe and record the cytopathic effect (CPE). CPE should occur in all virus control wells, and no CPE should appear in the neutralization wells and maintenance fluid control wells.

[0162] 2.3.4 Immunogenicity Adjust the virus content to 10 3.5 TCID 50 / 0.1 ml of the duck reovirus YBAH - 03 strain virus solution. After inactivating, prepare an inactivated vaccine according to the ratio of 1:2 (aqueous phase: oil phase). Use 20 healthy and susceptible 1 - to 2 - day - old breeding ducks. Inject 0.5 ml of the vaccine subcutaneously or intramuscularly into 10 of them, and use the other 10 as non - immunized controls. 15 - 16 days after immunization, inject the duck reovirus YBAH - 03 strain virus solution for challenge intramuscularly, 0.1 ml per duck (containing 10 5.0 TCID 50 ). Dissect on the 4th day after inoculation. The spleens of at least 8 control ducks should show varying degrees of swelling or necrosis; the spleens of at least 8 immunized ducks should not be swollen and not necrotic. Collect the spleens of the test ducks for virus isolation. At least 8 of the control ducks should be positive; at least 8 of the immunized ducks should be negative.

[0163] 2.3.5 Purity Conduct tests according to the appendix of the third part of the current "Chinese Veterinary Pharmacopoeia", the duck embryo fibroblast inspection method, and the duck hepatitis B virus inspection method. There should be no bacterial growth, no mycoplasma, and no exogenous virus contamination.

[0164] 2.3.6 Basic seed passage generations The basic seed passage generations for production are C6 - C8 generations; the basic seed passage generations for challenge are C4 - C6 generations.

[0165] 2.3.7 Virus strain preservation Preserve at - 70°C or below.

[0166] 2.4 Recombinant baculovirus Re - Bac VP2 strain for production

[0167] 2.4.1 Virus content Serial - dilute the virus strain 10 - fold with a commercial serum - free insect cell medium. Take 10 -4 、10 -5 、10 -6 、10 -7 4 dilution degrees, and inoculate them into a 96 - well culture plate with well - grown monolayer Sf9 insect cells. Inoculate 6 wells for each dilution degree, 0.1 ml per well. At the same time, set 6 wells as normal cell control wells. After culturing at 27°C for 96 hours, discard the culture medium, conduct an indirect immunofluorescence test, and calculate the TCID 50 according to the Reed - Muench method. The virus content per 0.1 ml should not be less than 10 6.0 TCID 50 .

[0168] 2.4.2 Protein content: In the bioreactor, the Hi5 insect cells were amplified and cultured to a cell density of 6.0×10 6 ~7.0×10 6 cells / ml, and then the cell density was diluted to 2.5×10 6 cells / ml with serum-free insect cell medium. The production strain of recombinant baculovirus Re-Bac VP2 was inoculated at an MOI of 0.4. The cells were cultured at 27°C with a dissolved oxygen value of 40%, and the stirring speed was adjusted to a suitable rotation speed matching the bioreactor. After culturing for 72 - 76 hours, the VP2 protein solution was harvested after standing and centrifugation. The VP2 protein content was detected and should not be less than 300 μg / ml, and the purity should not be less than 80%.

[0169] 2.4.3 Specificity

[0170] 2.4.3.1 PCR identification: The DNA of the virus strain was extracted for PCR detection, and a target band of about 210 bp should be amplified.

[0171] 2.4.3.2 Western Blot identification: The protein solution harvested in 2.4.2 was subjected to Western Blot, and a specific band should be detected at about 65 kDa.

[0172] 2.4.4 Immunogenicity: The protein solution harvested in 2.4.2 was adjusted to a target protein content of 20 μg / ml with sterile PBS solution, and inactivated to prepare an inactivated vaccine at a ratio of 1:2 (aqueous phase: oil phase). Twenty healthy and susceptible ducks at 1 - 2 days old were used, 10 ducks were each subcutaneously or intramuscularly injected with 0.5 ml of the vaccine in the neck, and 10 ducks were not immunized as a control. At 15 - 16 days after immunization, 0.2 ml (containing 10 5.5 ELD 50 ) of duck parvovirus YBFJ-02 strain was intramuscularly injected into the leg. Seven days after virus challenge, anal swabs were collected from all experimental ducks, and after treatment, PCR detection was performed. At least 8 ducks in the non-immunized and virus-challenged group should be PCR positive, and at least 8 ducks in the immunized group should be PCR negative.

[0173] 2.4.5 Purity: It was examined according to the appendix of the third part of the current "Chinese Veterinary Pharmacopoeia", and there should be no bacterial growth, no mycoplasma and exogenous virus contamination.

[0174] 2.4.6 Basic seed: Subgenerations F4 - F6.

[0175] 2.5 Duck parvovirus YBFJ-02 strain for virus challenge

[0176] 2.5.1 Virus content: The virus strain was serially diluted 10-fold with sterile PBS, and 10 -5 、10 -6 、10 -7 、10-8 Four dilution degrees, inoculate 5 susceptible duck embryos at 9 - 10 days old with 0.2 ml per embryo for each dilution degree on the chorioallantoic membrane; simultaneously, set up 5 susceptible duck embryos inoculated with sterile PBS as control, 0.2 ml per embryo. Incubate at 36 - 37 °C continuously, candling the embryos twice a day, and observe until 216 hours. Count the number of dead duck embryos and calculate the ELD 50 . The virus content per 0.2 ml should not be less than 10 6.0 ELD 50 .

[0177] 2.5.2 Virulence to duck embryos Dilute the virus strain appropriately with sterile PBS (about containing 200 ELD 50 / 0.2 ml), inoculate 10 susceptible duck embryos at 9 - 10 days old on the chorioallantoic membrane with 0.2 ml per embryo, incubate at 36 - 37 °C continuously, and observe until 216 hours. At least 8 duck embryos should die within 24 - 216 hours after inoculation, and the dead duck embryos should show the lesions of thickening and edema of the chorioallantoic membrane.

[0178] 2.5.3 Virulence to healthy susceptible ducklings Dilute the virus strain 10 - fold with sterile PBS, intramuscularly inject 10 healthy susceptible ducks at 16 - 18 days old with 0.2 ml per duck (containing 10 5.5 ELD 50 ), and simultaneously set up 5 ducks of the same batch without inoculation as control. Observe continuously for 7 days, collect anal swabs of all test ducks on the 7th day, and conduct PCR detection. All ducks in the control group should be healthy and alive and all PCR detections should be negative, and at least 8 ducks in the inoculation group should get sick (at least 8 are positive in PCR detection).

[0179] 2.5.4 Specificity Dilute the virus strain appropriately with sterile PBS (about containing 200 ELD 50 / 0.2 ml), mix it with an equal volume of anti - duck parvovirus positive serum, incubate at 37 °C for 1 hour for neutralization, then inoculate 10 susceptible duck embryos at 9 - 10 days old on the chorioallantoic membrane with 0.2 ml per embryo. At the same time, set up 10 virus controls, 0.1 ml per embryo (about containing 100 ELD 50 ), incubate at 36 - 37 °C continuously, and observe until 216 hours. Within 24 - 216 hours, at least 8 duck embryos in the neutralization group should survive; at least 8 duck embryos in the control group should die, and the dead duck embryos should show the lesions of thickening and edema of the chorioallantoic membrane.

[0180] 2.5.5 Purity Conduct inspections according to the appendix of Part III of the current "Chinese Veterinary Pharmacopoeia" and the inspection methods for duck embryo fibroblasts and duck hepatitis B virus. There should be no bacterial growth, no mycoplasma and no exogenous virus contamination.

[0181] 2.5.6 Basic seed passage generations The basic seed passage generations for production are E7 - E9 generations; the basic seed passage generations for virus challenge are E5 - E7 generations.

[0182] Storage of 2.6 strain: Store at a temperature below -70°C.

[0183] Example 3: Preservation period test of type 1 and type 3 duck hepatitis viruses at different temperatures

[0184] Store type 1 and type 3 duck hepatitis viruses separately at -20°C, -70°C, and liquid nitrogen. Measure the virus content and the median lethal dose for healthy susceptible ducks every 6 months (measured according to the aforementioned method).

[0185] Table 1: Results of the preservation period test of type 1 and type 3 duck hepatitis viruses at different temperatures ＊

[0186]

[0187] Note: 1. " / " indicates that this item was not carried out; 2. "*" indicates that when measuring the virus content and the median lethal dose for healthy susceptible ducks, the virus dilution gradient will be slightly adjusted according to the previous test results and the current results.

[0188] The results show that when type 1 and type 3 duck hepatitis viruses are stored below -20°C, the virus content and the median lethal dose for healthy susceptible ducks both decrease relatively quickly. When stored for 12 months, the virus content of type 1 duck hepatitis virus drops from 10 7.38 ELD 50 / 0.2 ml to 10 6.17 ELD 50 / 0.2 ml, and the median lethal dose for healthy susceptible ducks drops from 10 6.0 LD 50 / 0.5 ml to 10 4.83 LD 50 / 0.5 ml. The virus content of type 3 duck hepatitis virus drops from 10 7.00 ELD 50 / 0.2 ml to 10 5.83 ELD 50 / 0.2 ml, and the median lethal dose for healthy susceptible ducks drops from 10 5.83 LD 50 / 0.5 ml to 10 4.22 LD 50 / 0.5 ml. Compared with 0 months, both indicators decrease by more than 1 titer; when the two viruses are stored below -70°C and stored for 18 months, the virus content of type 1 duck hepatitis virus drops from 10 7.38 ELD 50 / 0.2 ml to 10 6.32 ELD 50 / 0.2 ml, and the median lethal dose for healthy susceptible ducks drops from 10 6.0 LD 50 / 0.5 ml to 104.75 LD 50 / 0.5 ml, the virus content of type 3 duck hepatitis virus decreased from 10 7.00 ELD 50 / 0.2 ml to 10 5.78 ELD 50 / 0.2 ml, the median lethal dose for healthy susceptible ducks decreased from 10 5.83 LD 50 / 0.5 ml to 10 4.32 LD 50 / 0.5 ml. Compared with 0 months, both indicators decreased by more than 1 titer, but the decrease rate was slightly slower than that of the data stored at below -20°C for 12 months. When 2 viruses were stored in liquid nitrogen until 36 months, the virus content of type 1 duck hepatitis virus decreased from 10 7.38 ELD 50 / 0.2 ml to 10 7.00 ELD 50 / 0.2 ml, the median lethal dose for healthy susceptible ducks decreased from 10 6.0 LD 50 / 0.5 ml to 10 5.75 LD 50 / 0.5 ml, the virus content of type 3 duck hepatitis virus decreased from 10 7.00 ELD 50 / 0.2 ml to 10 6.62 ELD 50 / 0.2 ml, the median lethal dose for healthy susceptible ducks decreased from 10 5.83 LD 50 / 0.5 ml to 10 5.50 LD 50 / 0.5 ml. Compared with 0 months, there was no obvious decrease in both indicators. Thus, it can be seen that type 1 and type 3 duck hepatitis viruses can be effectively stored in liquid nitrogen. After being stored for 36 months, their virus content and virulence did not change significantly. Longer storage period tests are still in progress.

[0189] Example 4 Source and Standards of Cell Lines for Production

[0190] 4.1 Source The original High five insect cell line was donated by East China University of Science and Technology. After being cloned and identified by Qingdao Yibang Bioengineering Co., Ltd. (hereinafter referred to as "the company"), a cell bank was established. The cell code is Hi5, which is kept and supplied by the company; the suspension LMH-C cells are cloned, domesticated, identified, kept and supplied by the company.

[0191] 4.2 Cell Line Standards

[0192] 4.2.1 Hi5 Cell Standards

[0193] 4.2.1.1 Morphology of Hi5 cells When cultured in serum-free insect cell medium and observed under a microscope, the cells should be in the shape of regular spheres, uniform in size, with good refractivity and dispersibility.

[0194] 4.2.1.2 Purity It shall be inspected according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia and should show no growth of bacteria, mycoplasma and exogenous virus contamination.

[0195] 4.2.1.3 Cell passage The passage of the basic cell bank is 3 - 6 passages, and the maximum passage number for production cells shall not exceed 20 passages.

[0196] 4.2.1.4 Preservation of cell seeds Preserved in liquid nitrogen.

[0197] 4.2.2 Standards for suspension LMH-C cells

[0198] 4.2.2.1 Morphology of suspension LMH-C cells When cultured in serum-free LMH medium, the suspension LMH-C cells with good growth are round, plump, translucent, uniform in size and clear in outline, with a viability of more than 90%.

[0199] 4.2.2.2 Purity It shall be inspected according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia and should show no growth of bacteria, mycoplasma and exogenous virus contamination.

[0200] 4.2.2.3 Cell passage The passage of the basic cell bank is 3 - 6 passages, and the maximum passage number for production cells shall not exceed 20 passages.

[0201] 4.2.2.4 Preservation of cell seeds Preserved in liquid nitrogen.

[0202] Preparation of main components in Example 5

[0203] 5.1 Preparation of production virus strains

[0204] 5.1.1 Preparation of strain YBH1 of type 1 duck hepatitis virus

[0205] 5.1.1.1 Virus strain propagation Dilute the virus strain 100 times and inoculate 0.2 ml into the allantoic cavity of susceptible chicken embryos at 10 - 11 days old. Incubate at 36 - 37 °C and candling the embryos twice a day.

[0206] 5.1.1.2 Harvest Harvest the chicken embryos that died within 48 - 120 hours after inoculation. Place them at 2 - 8 °C for 12 - 16 hours, then collect the allantoic fluid and embryo bodies. Homogenize, freeze-thaw 3 times, centrifuge at 4000 r / min for 30 minutes, and take the supernatant and mix it in a sterile container, indicating the harvest date and virus strain passage.

[0207] 5.1.1.3 Virus strain identification Conducted according to items 2.1.1, 2.1.5 and 2.1.7, and should meet the requirements.

[0208] 5.1.1.4 Strain preservation: Store at below -70°C for no more than 6 months.

[0209] 5.1.1.5 Subculture of the strain: Do not exceed 3 passages.

[0210] 5.1.2 Preparation of strain of Duck hepatitis A virus type 3 YBH3

[0211] 5.1.2.1 Strain propagation: Dilute the strain 100-fold, inoculate 10 - 11-day-old susceptible chicken embryos via the allantoic cavity at 0.2 ml per embryo, incubate at 36 - 37°C, and candicate the embryos twice a day.

[0212] 5.1.2.2 Harvest: For chicken embryos that die within 48 - 120 hours after inoculation, place them at 2 - 8°C for 12 - 16 hours, then collect the allantoic fluid and embryo bodies. After homogenization and 3 cycles of freezing and thawing, centrifuge at 4000 r / min for 30 minutes, take the supernatant and mix it in a sterile container, indicating the harvest date and strain passage number.

[0213] 5.1.2.3 Strain identification: Conduct according to items 2.2.1, 2.2.5 and 2.2.7, and it should comply with the regulations.

[0214] 5.1.2.4 Strain preservation: Store at below -70°C for no more than 6 months.

[0215] 5.1.2.5 Subculture of the strain: Do not exceed 3 passages.

[0216] 5.1.3 Preparation of strain of Duck reovirus YBAH - 03

[0217] 5.1.3.1 Select well - growing LMH - C adherent cells, discard the original culture medium, add the maintenance medium containing 1% of the strain, incubate at 37°C for 36 - 48 hours, harvest when the cytopathic effect reaches over 80%, freeze - thaw twice, aliquot into sterilized containers, indicate the harvest date, strain passage number, etc., and store at below -70°C.

[0218] 5.1.3.3 Strain identification: Conduct according to items 2.3.1, 2.3.3, 2.3.5, and it should comply with the regulations.

[0219] 5.1.3.4 Subculture of the strain: Should not exceed 3 passages.

[0220] 5.1.3.5 Strain preservation: At below -70°C for no more than 12 months.

[0221] 5.1.4 Preparation of strain of Recombinant baculovirus Re - Bac VP2

[0222] 5.1.4.1 Strain Propagation Take the recombinant baculovirus Re-Bac VP2 strain, inoculate the Sf9 insect cells cultured in suspension at a ratio of 1.0% of the cell culture volume, culture at 27°C for 72 hours, harvest the culture, freeze-thaw it 3 times repeatedly, quantitatively sub-pack, mark the strain name, passage number, harvest date, etc., and use it as the production strain, store it below -70°C.

[0223] 5.1.4.2 Strain Identification Conduct according to items 2.1.1, 2.1.3 and 2.1.5, and it should meet the requirements.

[0224] 5.1.4.3 Strain Preservation Store below -70°C, and the storage period should not exceed 12 months.

[0225] 5.1.4.4 Strain Subculture The subculture should not exceed 3 passages.

[0226] 5.2 Selection of Vaccine Preparation Materials

[0227] 5.2.1 Duck Hepatitis Virus Vaccine Preparation Materials For Duck Hepatitis Virus Type 1 YBH1 strain and Duck Hepatitis Virus Type 3 YBH3 strain, well-developed 10 - 11-day-old susceptible chicken embryos are selected as vaccine preparation materials.

[0228] 5.2.2 Duck Reovirus Vaccine Preparation Materials LMH-C suspension cells and suspension serum-free LMH cell culture medium are used as vaccine preparation materials.

[0229] 5.2.3 Novel Duck Parvovirus Vaccine Preparation Materials Hi5 insect cells and insect cell serum-free culture medium are used as vaccine preparation materials.

[0230] 5.3 Preparation of Antigen Solution for Vaccine Preparation

[0231] 5.3.1 Duck Hepatitis Virus Type 1 Antigen

[0232] 5.3.1.1 Inoculation Take the production strain, dilute it 100 times with sterile PBS, inoculate the allantoic cavity of 10 - 11-day-old susceptible chicken embryos, 0.2 ml per embryo, and continue to incubate at 36 - 37°C.

[0233] 5.3.1.2 Incubation and Observation After inoculating the chicken embryos, candling the embryos twice a day. For the chicken embryos that die within 48 - 120 hours after inoculation, place them at 2 - 8°C for 12 - 16 hours.

[0234] 5.3.1.3 Harvest Take out the cooled chicken embryos, collect the allantoic fluid and embryo bodies. After homogenization, freeze-thaw 3 times, centrifuge at 4000 r / min for 30 minutes, take the supernatant and mix it in a sterile container, keep a sample for semi-finished product inspection, and inactivate the remaining embryo fluid immediately.

[0235] 5.3.1.4 Inactivation Introduce the virus solution into the inactivation tank, measure and add 10% formaldehyde solution, mix well, and the final concentration of the formaldehyde solution is 0.2%. After inactivating at 37°C for 16 hours (starting from when the temperature in the tank reaches 37°C), take it out and store it at 2 - 8°C, and it should not exceed 30 days.

[0236] 5.3.2 Duck hepatitis A virus type 3 antigen

[0237] 5.3.2.1 Inoculation Take the production strain, dilute it 100 - fold with sterile PBS, and inoculate the allantoic cavity of 10 - 11-day-old susceptible chicken embryos, 0.2 ml per embryo, and continue to incubate at 36 - 37°C.

[0238] 5.3.2.2 Incubation and observation After inoculating the chicken embryos, candling the embryos twice a day. For the chicken embryos that die within 48 - 120 hours after inoculation, place them at 2 - 8°C for 12 - 16 hours.

[0239] 5.3.2.3 Harvest Take out the cooled chicken embryos, collect the allantoic fluid and embryo bodies. After homogenization, freeze-thaw 3 times, centrifuge at 4000 r / min for 30 minutes, take the supernatant and mix it in a sterile container, keep a sample for semi-finished product inspection, and inactivate the remaining embryo fluid immediately.

[0240] 5.3.2.4 Inactivation Introduce the virus solution into the inactivation tank, measure and add 10% formaldehyde solution, mix well, and the final concentration of the formaldehyde solution is 0.2%. After inactivating at 37°C for 16 hours (starting from when the temperature in the tank reaches 37°C), take it out and store it at 2 - 8°C, and it should not exceed 30 days.

[0241] 5.3.3 Duck reovirus antigen

[0242] 5.3.3.1 Preparation of production cells

[0243] 5.3.3.1.1 Cell resuscitation Resuscitate the cryopreserved suspension LMH-C cells from liquid nitrogen by the conventional method, add the cell suspension to a triangular shaking flask with a pipette, and add a filter-sterilized serum-free medium with a pH value of 7.0 ± 0.2. Place the shaking flask in a shaker and culture and expand it at 37°C and 120 rpm. When the cell quantity is sufficient, it can be inoculated into a bioreactor for culture.

[0244] 5.3.3.1.2 Scale-up culture First, proliferate the suspension LMH-C cells, inoculate them into the bioreactor for culture at a density of about 1.8×10 6.0 ~2.0×10 6.0 cells / ml, at a temperature of 37°C, dissolved oxygen of 40% - 50%, pH value of 7.0 ± 0.2, adjust the rotation speed for culture, take samples every 24 hours to observe the cell growth situation, and at the same time perform cell counting. When the cell density reaches 6.0×10 6.0 ~7.0×10 6.0When the cell density reaches [specific number] cells / ml, transfer the cells into a large reactor that has been pre-sterilized and pre-incubated with cell culture medium, and continue the scale-up culture.

[0245] 5.3.3.2 Preparation of Duck Reovirus Antigen

[0246] 5.3.3.2.1 Virus Inoculation When the cell density in the final reactor reaches 6.0×10 6.0 ~7.0×10 6.0 cells / ml, it can be used for virus inoculation and culture. Transfer the cell suspension into a reactor that has been pre-sterilized and pre-incubated with virus culture medium. After stirring evenly, when the cell density reaches 2.5×10 6.0 cells / ml, inoculate the cell poison for duck reovirus production at an MOI of 0.2. Adjust the appropriate rotation speed of the reactor, pH value of 7.2±0.2, dissolved oxygen of 40% - 50%, and temperature of 37℃±0.1℃ for virus culture.

[0247] 5.3.3.2.2 Observation Twenty-four hours after inoculation, take samples to observe cytopathic effects (cell swelling and fragmentation), and at the same time detect the cell viability.

[0248] 5.3.3.2.3 Harvest When the cell viability drops to 30% - 40%, harvest the virus solution and take samples for semi-finished product inspection. Store at below -15℃ for no more than 30 days.

[0249] 5.3.3.2.4 Inactivation Pour the virus solution into the inactivation tank, measure and add 10% formaldehyde solution, mix well, and the final concentration of the formaldehyde solution is 0.2%. After inactivating at 37℃ for 16 hours (starting from when the temperature in the tank reaches 37℃), take it out and store at 2 - 8℃ for no more than 30 days.

[0250] 5.3.4 Preparation of VP2 Protein Solution

[0251] 5.3.4.1 Proliferation of Hi5 Insect Cells Take Hi5 insect cells out of liquid nitrogen and melt them in a 37℃ water bath. Add them to a shaking flask containing serum-free medium for insect cells and culture them in a shaker at 27℃ with a rotation speed of 130 r / min. After 48 hours, when the cell density reaches 6.0×10 6 ~7.0×10 6 cells / ml, passage the cells. Then gradually scale up the culture to proliferate Hi5 insect cells until the number of cells reaches the number required for the bioreactor.

[0252] 5.3.4.2 Virus Culture When the cell density reaches 6.0×10 6 ~7.0×10 6 cells / ml, dilute the cell density to 2.5×10 6Cells / ml, inoculate the production strain of recombinant baculovirus Re-Bac VP2 at an MOI of 0.4. Incubate at 27°C with a dissolved oxygen value of 40%, adjust the stirring speed to the rotation speed matching the bioreactor, and incubate for 90 - 94 hours.

[0253] 5.3.4.3 Extraction of VP2 protein solution After the incubation, let it stand at 2 - 8°C for 72 hours, centrifuge to remove cell debris, and the harvested supernatant is the VP2 protein solution. Take samples for determination of protein content and purity of the target protein. Store at 2 - 8°C, and the storage period should not exceed 30 days.

[0254] 5.3.4.4 Inactivation of VP2 protein solution Slowly add 10% formaldehyde solution to the VP2 protein solution, mix well, so that the final concentration of formaldehyde solution is 0.1%, inactivate at 37°C for 48 hours (starting from the time when the temperature in the tank reaches 37°C, stir once every 6 hours during this period), then take out, take samples for sterility test and inactivation test. Store at 2 - 8°C, and the storage period should not exceed 30 days.

[0255] 6 Semi-finished product inspection

[0256] 6.1 Duck viral hepatitis part

[0257] 6.1.1 Determination of virus content

[0258] 6.1.1.1 Determination of virus content of YBH1 strain Take the concentrated virus solution of YBH1 strain, and determine it according to the method in 2.1.1. The virus content per 0.2 ml should not be less than 10 6.5 ELD 50 。

[0259] 6.1.1.2 Determination of virus content of YBH3 strain Take the concentrated virus solution of YBH3 strain, and determine it according to the method in 2.2.1. The virus content per 0.2 ml should not be less than 10 6.5 ELD 50 。

[0260] 6.1.2 Sterility test Take the inactivated concentrated virus solution and conduct the test according to the appendix of Part III of the current "Chinese Veterinary Pharmacopoeia", and there should be no bacterial growth.

[0261] 6.1.3 Inactivation test

[0262] 6.1.3.1 Inactivation test of YBH1 strain virus solution Inoculate 10 susceptible chicken embryos at 10 - 11 days old with 0.2 ml of the inactivated virus solution of YBH1 strain into the allantoic cavity, incubate at 36 - 37°C, observe continuously for 168 hours, and the non-specific death of chicken embryos (death caused by inoculation or weak embryos within 24 hours, the same below) should not exceed 1. Blind passage once according to this method, and if the non-specific death of chicken embryos after blind passage does not exceed 1, it is judged that the inactivation test is qualified.

[0263] 6.1.3.2 Inactivation Test of YBH3 Strain Virus Liquid Inoculate 10 susceptible chicken embryos at 10 - 11 days old with 0.2 ml of the inactivated virus liquid of YBH3 strain into the allantoic cavity, incubate at 36 - 37 °C, continuously observe for 168 hours. The non - specific death of chicken embryos should not exceed 1. Blind - passage once according to this method. If the non - specific death of chicken embryos after blind - passage does not exceed 1, it is judged that the inactivation test is qualified.

[0264] 6.2 Duck Reovirus Part

[0265] 6.2.1 Virus Content Determination Take the virus liquid of YBAH - 03 strain before inactivation and measure it according to the method in 2.3.1 (since this virus is cultured without serum using suspension LMH - C cells, the virus content is much higher than that cultured with LMH - C adherent cells for the virus seed. Therefore, when using the method in 2.3.1 for virus content determination, the dilution factors should be adjusted to 10 -6 、10 -7 、10 -8 、10 -9 4 dilution factors, and the others are the same as the method in 2.3.1). The virus content per 0.1 ml should not be less than 10 8.0 TCID 50 。

[0266] 6.2.2 Sterility Test Take the inactivated virus liquid and test it according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia. There should be no bacterial growth.

[0267] 6.2.3 Inactivation Test Dilute the inactivated virus liquid 10 - fold, inoculate 4 wells of well - grown LMH - C adherent cells (24 - well cell plate) with 0.2 ml per well, and supplement the maintenance medium to 2.0 ml; at the same time, set up non - inoculated LMH - C adherent cells as a blank control, incubate in an incubator at 37 °C and 5% CO 2 for 168 hours. There should be no cytopathic effect in both the cell control wells and the sample wells. Harvest the culture, freeze - thaw repeatedly, and then blind - passage once, continue to culture for 168 hours. When there is still no cytopathic effect in the sample wells, it is judged that the inactivation is complete.

[0268] 6.3 VP2 Antigen Part

[0269] 6.3.1 VP2 Protein Content Determination Take the virus liquid of the recombinant baculovirus Re - Bac VP2 strain before inactivation and measure the VP2 protein content according to the Bradford method. It should not be less than 300 μg / ml, and the purity should not be less than 80%.

[0270] 6.3.2 Sterility Test Take the inactivated virus liquid and test it according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia. There should be no bacterial growth.

[0271] 6.3.3 Inactivation Test Inoculate the inactivated VP2 antigen solution into a 25 cm cell flask with well-grown monolayer Sf9 insect cells at a volume ratio of 1.0%, culture at 27 °C for 96 hours, harvest the culture medium and freeze-thaw it 3 times, and blindly passage it for 2 generations in this way. Inoculate the finally harvested culture medium into a 96-well cell plate with well-grown monolayer Sf9 insect cells at a volume ratio of 1.0%. At the same time, take the non-inactivated antigen solution and inoculate it at a volume ratio of 1.0% as a positive control, and normal Sf9 insect cells as a negative control. Each of the test sample, positive, and negative controls is inoculated into 6 wells. After continuing to culture for 96 hours, perform an indirect immunofluorescence test. Specific fluorescence should appear in the positive control wells, no specific fluorescence should appear in the negative control wells, and the test sample wells are judged to be qualified for inactivation test if no specific fluorescence appears. 2 Cell flask, culture at 27 °C for 96 hours, harvest the culture medium and freeze-thaw it 3 times, and blindly passage it for 2 generations in this way. Inoculate the finally harvested culture medium into a 96-well cell plate with well-grown monolayer Sf9 insect cells at a volume ratio of 1.0%. At the same time, take the non-inactivated antigen solution and inoculate it at a volume ratio of 1.0% as a positive control, and normal Sf9 insect cells as a negative control. Each of the test sample, positive, and negative controls is inoculated into 6 wells. After continuing to culture for 96 hours, perform an indirect immunofluorescence test. Specific fluorescence should appear in the positive control wells, no specific fluorescence should appear in the negative control wells, and the test sample wells are judged to be qualified for inactivation test if no specific fluorescence appears.

[0272] Example 6 Vaccine Preparation

[0273] 7.1 Preparation

[0274] 7.1.1 Preparation of Oil Phase Take 96 parts of mineral oil and 0.67 parts of aluminum stearate, mix them evenly in an oil phase preparation tank and heat to 80 °C, then add 4 parts of Span-80. When the temperature reaches 115 °C, maintain for 40 minutes, and cool for later use.

[0275] 7.1.2 Preparation of Aqueous Phase Dilute the qualified antigen solution appropriately so that the virus content of type 1 duck hepatitis virus and type 3 antigen is 10 ELD50 / 0.2 ml, and the virus content of duck reovirus antigen is 10 TCID50 / ml. Mix them in a 1:1:1 ratio, and add duck parvovirus VP2 protein solution to make its final concentration 40 μg / ml. Take 96.6 parts of the mixed antigen solution and 3.4 parts of sterile Tween-80, mix well to completely dissolve Tween-80. 6.5 ELD 50 / 0.2 ml, and the virus content of duck reovirus antigen is 10 5.5 TCID 50 / ml. Mix them in a 1:1:1 ratio, and add duck parvovirus VP2 protein solution to make its final concentration 40 μg / ml. Take 96.6 parts of the mixed antigen solution and 3.4 parts of sterile Tween-80, mix well to completely dissolve Tween-80.

[0276] Take 96.6 parts of the mixed antigen solution and 3.4 parts of sterile Tween-80, mix well to completely dissolve Tween-80.

[0277] 7.1.3 Emulsification Put 2 parts of the oil phase into an emulsification tank, start the motor, rotate the stirrer slowly, and while slowly adding 1 part of the aqueous phase, then stir at 3500 r / min for 30 - 40 minutes. After emulsification, take 10 ml of the vaccine and add it to a centrifuge tube, centrifuge at 3000 r / min for 15 minutes, and the water phase separated at the bottom of the tube should not exceed 0.5 ml.

[0278] 7.1.4 Sub-packaging Quantitatively sub-package according to the specification requirements, cover and seal.

[0279] 7.2 Final Product Factory Inspection

[0280] 7.2.1 Characters

[0281] Appearance: Milky white and uniform emulsion.

[0282] Dosage form: Water-in-oil type. Take a clean pipette, suck a small amount of the vaccine and drop it on the surface of cold water. Except for the first drop, it should not spread.

[0283] Stability: Take 10 ml of the vaccine and add it to a centrifuge tube. Centrifuge at 3000 r / min for 15 minutes. The water separated at the bottom of the tube should not exceed 0.5 ml.

[0284] Viscosity: Examine according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia and should comply with the regulations.

[0285] 7.2.2 Inspection of filling quantity: Inspect according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia and should comply with the regulations.

[0286] 7.2.3 Sterility test: Examine according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia and there should be no growth of bacteria.

[0287] 7.2.4 Safety test: Use 10 healthy and susceptible ducks at 3 to 5 days old. Inject 1.0 ml of the vaccine subcutaneously or intramuscularly into the neck of each duck. Observe for 14 days. There should be no local or systemic adverse reactions caused by the vaccine.

[0288] 7.2.5 Potency test

[0289] 7.2.5.1 For duck hepatitis virus, either of the following two methods can be chosen.

[0290] 7.2.5.1.1 Serological method: Use 15 healthy and susceptible ducks at 21 to 35 days old. Inject 0.5 ml of the vaccine subcutaneously or intramuscularly into the neck of 10 ducks, and 5 ducks are not immunized as controls. 21 to 28 days after immunization, collect blood from each duck to separate serum, and measure the neutralizing antibody titers of type 1 and type 3 duck hepatitis viruses respectively. The antibody titers of the immunized group should not be lower than 1:40, and those of the control group should all be negative.

[0291] 7.2.5.1.2 Immunization and challenge method: Use 40 healthy and susceptible ducks at 1 to 2 days old. Inject 0.5 ml of the vaccine subcutaneously or intramuscularly into the neck of 20 ducks, and 20 ducks are not immunized as controls. 15 to 16 days after immunization, take 10 ducks from the immunized group and the control group respectively, and inject 0.5 ml (containing 10 4.5 LD 50 ) of duck hepatitis virus strain YBH1 into the leg muscle; inject 0.5 ml (containing 10 4.5 LD 50 ) of duck hepatitis virus strain YBH3 into the leg muscle of the other 20 ducks. Observe for 7 days after challenge. At least 8 ducks in the immunized group should survive healthily, and at least 8 ducks in the control group should die.

[0292] 7.2.5.2 For duck reovirus, 20 healthy and susceptible ducks at 1 - 2 days old were used. 10 ducks were each subcutaneously or intramuscularly injected with 0.5 ml of the vaccine in the neck, and 10 ducks were not immunized as controls. 15 - 16 days after immunization, the ducks were respectively intramuscularly injected with the virus solution of duck reovirus strain YBAH - 03 for challenge, 0.1 ml per duck (containing 10 5.0 TCID 50 ). The ducks were sacrificed on the 4th day after inoculation. The spleens of at least 8 control ducks should show varying degrees of swelling or necrosis; the spleens of at least 8 immunized ducks should not be swollen and not necrotic. The spleens of the test ducks were collected for virus isolation. At least 8 control ducks should be positive; at least 8 immunized ducks should be negative.

[0293] 7.2.5.3 For duck parvovirus, 20 healthy and susceptible ducks at 1 - 2 days old were used. 10 ducks were each subcutaneously or intramuscularly injected with 0.5 ml of the vaccine in the neck, and 10 ducks were not immunized as controls. 15 - 16 days after immunization, the ducks were respectively intramuscularly injected with 0.2 ml of duck parvovirus strain YBFJ - 02 in the leg (containing 10 5.5 ELD 50 ). 7 days after challenge, anal swabs were collected from all test ducks, and after processing, PCR detection was carried out. At least 8 ducks in the non - immunized challenged group should be PCR - positive, and at least 8 ducks in the immunized group should be PCR - negative.

[0294] Example 8 Vaccine Safety and Efficacy Testing

[0295] 8.1 Safety test results of three batches of inactivated vaccines against duck viral hepatitis (type 1 + type 3), novel duck reovirus disease, and novel duck parvovirus disease (abbreviated as "quadrivalent combined vaccine") The safety tests of 3 batches of quadrivalent combined vaccines were carried out according to the method in item 7.2.4. After inoculating 3 - day - old ducklings with the 3 batches of vaccines, during the observation period, no local or systemic adverse reactions occurred in all test ducks, and there were no obvious abnormalities in spirit, food intake, water intake, respiration, feces, and hair. At 14 days after inoculation, during necropsy, the inoculation sites were well - absorbed, and there were no gross lesions such as redness, ulceration, nodules, and granulomas.

[0296] Table 2 Safety test results of three batches of vaccines

[0297]

[0298]

[0299] Note: "-" indicates no adverse reaction.

[0300] 8.2 Efficacy test results of three batches of quadrivalent combined vaccines

[0301] 8.2.1 Efficacy test for duck hepatitis part

[0302] 8.2.1.1 Serological method The serological method (serum neutralizing antibody titer) was used to detect the duck hepatitis part of 3 batches of triple-valent and tetravalent vaccines according to the method described in item 7.2.5.1.1. Twenty-one-day-old ducklings were immunized with the vaccine, and blood was collected 21 days after immunization. Serum was separated, and the serum neutralizing antibody titers against type 1 and type 3 duck hepatitis viruses were measured respectively. The geometric mean titers of the serum neutralizing antibody against type 1 duck hepatitis virus in the 3 batches of vaccines were 1:136, 1:105, and 1:119 respectively. The lowest individual neutralizing antibody titer was 1:90, and the highest was 1:149. The dispersion of the geometric mean titers of the serum neutralizing antibody against type 1 duck hepatitis virus in the 3 batches of vaccines was not significant. The geometric mean titers of the serum neutralizing antibody against type 3 duck hepatitis virus were 1:119, 1:89, and 1:106 respectively. The lowest individual neutralizing antibody titer was 1:40, and the highest was 1:131. The dispersion of the geometric mean titers of the serum neutralizing antibody against type 3 duck hepatitis virus in the 3 batches of vaccines was not significant. The serum neutralizing antibody titers of the control ducks against type 1 and type 3 duck hepatitis viruses were both negative (both 5 / 5 ≤ 1:2). See Table 3 for details.

[0303] Table 3 Results of the potency test of the duck hepatitis part of three batches of vaccines (serum neutralizing antibody titer)

[0304]

[0305]

[0306] Note: " / " indicates that this item was not carried out. (The same below)

[0307] 8.2.1.2 Immunization and challenge method The potency test of the duck hepatitis part of 3 batches of triple-valent and tetravalent vaccines was carried out by the immunization and challenge method according to the method described in item 7.2.5.1.2. The protection rates of the 3 batches of vaccines against type 1 duck hepatitis virus were all 100% (10 / 10 protected) when challenged 16 days after immunization. After challenge, the mental state, food intake, water intake, respiration, feces, and hair of the immunized ducks were all normal. Three of the control ducks died 24 hours after being challenged with type 1 duck hepatitis virus, 4 died 36 hours later, 2 died 48 hours later, and 1 died 60 hours later, and all died within 7 days of the observation period (10 / 10 died). The protection rates of the immunized ducks against type 3 duck hepatitis virus were 90% and above 7 days after challenge (only 9 / 10 protected for batch 01, and 10 / 10 protected for batches 02 and 03). Only 1 experimental duck in batch 01 died 84 hours after being challenged with type 3 duck hepatitis virus, and symptoms of duck hepatitis virus infection were found during autopsy. The mental state, food intake, water intake, respiration, feces, and hair of the remaining immunized ducks were all normal. Two of the control ducks died 24 hours after being challenged with type 3 duck hepatitis virus, 4 died 36 hours later, 1 died 48 hours later, and 2 died 60 hours later, and 9 died within 7 days of the observation period (9 / 10 died). The remaining 1 duck showed a slightly poor mental state and was reluctant to move 48 to 60 hours after being challenged, and then returned to normal. See Table 4 for details.

[0308] Table 4 Partial potency test results of three batches of duck hepatitis vaccine (immunization and challenge)

[0309]

[0310]

[0311] 8.2.2 Partial potency test of duck reovirus The partial potency test of duck reovirus for three batches of triple quadrivalent vaccine was carried out according to the method in item 7.2.5.2. After immunizing 2-day-old ducklings with three batches of vaccine, they were challenged with strain YBAH-03 on the 16th day. During the observation period, all test ducks had no adverse reactions. Four days after challenge, autopsy was performed. The results showed that the spleens of immunized ducks were all normal, and no splenomegaly or necrotic patches were seen; after grinding and processing the spleens for virus isolation, no virus was isolated; after autopsy of control ducks, 9 / 10 spleens were enlarged with necrotic patches of different sizes, and after grinding and processing the spleens for virus isolation, 9 / 10 were positive for virus isolation. It shows that the protection rate of the three batches of vaccines against the new reovirus virulent strain is 100% (10 / 10 protection). See Table 5 for details.

[0312] Table 5 Partial potency test results of three batches of vaccine against new duck reovirus disease

[0313]

[0314] Note: 1. In the spleen lesion item, "-" indicates that no necrosis or enlargement was seen in the dissected spleen; "+" indicates that necrosis or enlargement was seen in the dissected spleen.

[0315] 2. In the virus isolation item, "-" indicates negative virus isolation, that is, no new duck reovirus was isolated; "+" indicates positive virus isolation, that is, new duck reovirus was isolated.

[0316] 8.2.3 Partial potency test of duck parvovirus The partial potency test of duck parvovirus for three batches of triple quadrivalent vaccine was carried out according to the method in item 7.2.5.3. After immunizing 2-day-old ducklings with three batches of vaccine, they were challenged with strain YBFJ-02 on the 16th day. During the observation period, all test ducks had no adverse reactions. Seven days after challenge, anal swabs were collected from all test ducks and processed for PCR detection. The results showed that only 1 duck in batch 03 of the immunized ducks was detected positive for duck parvovirus (1 / 10 PCR positive), and the rest were negative for PCR detection, and the vaccine protection rate was 90% and above; while 9 / 10 of the anal swab PCR detections of the control group were positive 7 days after challenge. It shows that after immunizing with this vaccine, the virus excretion of test ducks can be effectively inhibited. See Table 6 for details.

[0317] Table 6 Partial potency test results of three batches of vaccine against new duck parvovirus disease

[0318]

[0319]

[0320] Note: "-" indicates a negative PCR test, indicating that novel duck parvovirus was not detected; "+" indicates a positive PCR test, indicating that novel duck parvovirus was detected.

[0321] Example 9 Comparison of the triple quadrivalent inactivated vaccine against duck viral hepatitis (type 1 + type 3), novel duck reovirus disease, and novel duck parvovirus disease with similar products (since there are currently no commercially available inactivated vaccines for novel reovirus disease and novel duck parvovirus disease, only comparison with the bivalent duck hepatitis vaccine is made)

[0322] 9.1 Safety comparison: Twenty 3- to 5-day-old healthy susceptible ducks were used. Ten ducks were subcutaneously or intramuscularly injected with 1.0 ml of the triple quadrivalent inactivated vaccine against duck viral hepatitis (type 1 + type 3), novel duck reovirus disease, and novel duck parvovirus disease (abbreviated as "triple quadrivalent vaccine") in the neck. Another ten ducks were subcutaneously or intramuscularly injected with 1.0 ml of the duck hepatitis (type 1 + type 3) vaccine (abbreviated as "duck hepatitis bivalent vaccine") in the neck. Observation was carried out for 14 days, and whether there were local or systemic adverse reactions in the test ducks was recorded.

[0323] Table 7 Results of the safety comparison test between the triple quadrivalent vaccine and the duck hepatitis bivalent vaccine

[0324]

[0325] Note: "-" indicates no adverse reaction.

[0326] The results are shown in Table 7. After inoculating 3-day-old ducklings with the two vaccines, both were safe. During the observation period, no local or systemic adverse reactions occurred in the test ducks inoculated with the two vaccines. At 14 days after inoculation, autopsy showed good absorption at the inoculation site, and there were no gross lesions such as redness, ulceration, nodules, and granulomas.

[0327] 9.2 Immunogenicity comparison

[0328] 9.2.1 Serological method: Twenty-five 21- to 35-day-old SPF ducks were used. Ten ducks were subcutaneously or intramuscularly injected with 0.5 ml of the triple quadrivalent vaccine in the neck. Another ten ducks were subcutaneously or intramuscularly injected with 0.5 ml of the duck hepatitis bivalent vaccine in the neck. The remaining five ducks were not immunized as controls. At 21 to 28 days after immunization, blood was collected from each duck to separate serum, and the neutralizing antibody titers against duck hepatitis virus type 1 and type 3 were measured respectively.

[0329] Table 8 Results of the immunogenicity comparison test between the triple quadrivalent vaccine and the duck hepatitis bivalent vaccine (serum neutralizing antibody titers)

[0330]

[0331] The results are shown in Table 8. On the 21st day after immunization with the triple-valent tetravalent vaccine, the geometric mean of the serum neutralizing antibody titer against Duck hepatitis A virus type 1 was 1:105, and the lowest neutralizing antibody titer of 10 test ducks was 1:90. The geometric mean of the serum neutralizing antibody titer against Duck hepatitis A virus type 3 was 1:89, and the lowest neutralizing antibody titer of 10 test ducks was 1:40. On the 21st day after immunization with the bivalent duck hepatitis vaccine, the geometric mean of the serum neutralizing antibody titer against Duck hepatitis A virus type 1 was 1:83, and the lowest neutralizing antibody titer of 10 test ducks was 1:57. The geometric mean of the serum neutralizing antibody titer against Duck hepatitis A virus type 3 was 1:60, and the lowest neutralizing antibody titer of 10 test ducks was 1:24. From the perspective of the neutralizing antibody level after immunization, the antibody level of the triple-valent tetravalent vaccine was significantly higher than that of the commercially available bivalent duck hepatitis vaccine.

[0332] 9.2.2 Immunization and challenge method: Use 60 healthy and susceptible ducks at 1 - 2 days old. Take 20 ducks and inject 0.5 ml of the triple-valent tetravalent vaccine subcutaneously or intramuscularly into the neck of each. Take another 20 ducks and inject 0.5 ml of the bivalent duck hepatitis vaccine subcutaneously or intramuscularly into the neck of each. The remaining 20 ducks are not immunized as controls. On the 15th - 16th day after immunization, take 10 ducks from each of the immunized group and the control group, and inject 0.5 ml (containing 10 4.5 LD 50 ) of Duck hepatitis A virus strain YBH1 intramuscularly into the leg. For the other 30 ducks, inject 0.5 ml (containing 10 4.5 LD 50 ) of Duck hepatitis A virus strain YBH3 intramuscularly into the leg. Observe for 7 days after challenge, and record the death situation of the test ducks in the immunized group and the control group.

[0333] Table 9 Comparison of the efficacy of the triple-valent tetravalent vaccine and the bivalent duck hepatitis vaccine (immunization and challenge)

[0334]

[0335]

[0336] The results are shown in Table 9. Sixteen days after immunization with the triple-valent quadrivalent vaccine and the duck hepatitis bivalent vaccine, half of each immunized group was challenged with Duck hepatitis A virus type 1 strain YBH1. The protection rate of the triple-valent quadrivalent vaccine against strain YBH1 was 100% (10 / 10 protected), and the protection rate of the commercially available duck hepatitis bivalent vaccine against strain YBH1 was 80% (8 / 10 protected), with 20% mortality (2 / 10 died). After the control group was challenged with Duck hepatitis A virus type 1 strain YBH1, 100% of them developed the disease (10 / 10 died). The other half of the immunized group was challenged with Duck hepatitis A virus type 3 strain YBH3. The protection rate of the triple-valent quadrivalent vaccine against strain YBH3 was 90% (9 / 10 protected), with 10% mortality (1 / 10 died). The protection rate of the commercially available duck hepatitis bivalent vaccine against strain YBH3 was 80% (8 / 10 protected), with 20% mortality (2 / 10 died). After the control group was challenged with Duck hepatitis A virus type 3 strain YBH3, 90% of them developed the disease (9 / 10 died). Judging from the immune challenge protection effect, the protection effect of the triple-valent quadrivalent vaccine is better than that of the commercially available duck hepatitis bivalent vaccine.

Claims

1. A triple quadrivalent inactivated vaccine for preventing and treating duck viral diseases, characterized in that: The aqueous phase was prepared by mixing duck hepatitis virus type 1 antigen solution, duck hepatitis virus type 3 antigen solution and duck reovirus antigen solution, and then adding duck parvovirus VP2 protein solution to obtain a mixed antigen solution with a final concentration of 40 μg / ml, wherein the virus content in duck hepatitis virus type 1 antigen solution and duck hepatitis virus type 3 antigen solution was 10 6.5 ELD 50 / 0.2ml, the virus content of duck reovirus antigen is 10 5.5 TCID 50 / ml.

2. The triple quadrivalent inactivated vaccine according to claim 1, characterized in that: The volume ratio of duck hepatitis virus type 1 antigen solution, duck hepatitis virus type 3 antigen solution and duck reovirus antigen solution added to the aqueous phase is 1:1:

1.

3. The triple quadrivalent inactivated vaccine according to claim 1 or 2, characterized in that: The duck hepatitis virus type 1 strain is the YBH 1 strain, which was deposited in the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCCNO: V202513; The duck hepatitis virus type 3 strain is the YBH 3 strain, which was deposited in the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: V202514; The duck reovirus strain is the YBAH-03 strain, which was deposited in the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: V202511; The recombinant baculovirus Re-Bac VP2 strain expressing duck parvovirus VP2 protein was deposited in the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: V202512.

4. The triple quadrivalent inactivated vaccine according to claim 3, characterized in that: Duck reovirus was propagated by inoculation on chicken hepatoma suspension cells LMH-C. The viral content of the propagated duck reovirus was 10 8.5 TCID 50 / 0.1ml, when preparing the vaccine, dilute it to 10 5.5 TCID 50 / ml for use.

5. The triple quadrivalent inactivated vaccine according to claim 4, characterized in that: The suspension cell LMH-C was deposited in the China Center for Type Culture Collection in Wuhan on February 20, 2025, with the deposit number CCTCC NO: C202558.

6. The triple quadrivalent inactivated vaccine according to any one of claims 1 to 5, characterized in that The oil phase is prepared by uniformly mixing 96±2 parts of mineral oil and 0.67 parts of aluminum stearate, heating the mixture to 80°C±5°C, adding 4±2 parts of Siben-80, and maintaining the mixture at 115°C±5°C for 40±5 minutes, and then cooling the mixture to obtain the oil phase.

7. The triple quadrivalent inactivated vaccine according to claim 6, characterized in that: When the oil phase and the water phase are emulsified, the volume ratio of the two is 2:

1. Before emulsification, 96.6±2 parts of the mixed antigen solution and 3.4±2 parts of sterile Tween-80 of the water phase are fully mixed to completely dissolve the Tween-80.

8. Use of the triple quadrivalent inactivated vaccine according to any one of claims 1 to 7 in the simultaneous prevention and treatment of duck hepatitis virus type 1, duck hepatitis virus type 3, new duck reovirus disease, and new duck parvovirus disease.

9. The use according to claim 8, characterized in that: Healthy susceptible ducks of 1 to 2 days old were immunized by injecting 0.5 ml of triple quadrivalent inactivated vaccine subcutaneously or intramuscularly in the neck.

10. The use according to claim 8, characterized in that: After immunization, ducks were protected 10 / 10 against type 1 duck hepatitis, 9 / 10 and above against type 3 duck hepatitis, and the serum neutralizing antibody titer was not less than 1:40; ducks were protected 10 / 10 against the new reovirus test; ducks were protected 9 / 10 and above against the new duck parvovirus test.

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