Pigeon adenovirus SP strain and its application

Abstract

The present invention relates to the field of viruses, and in particular to a pigeon adenovirus SP strain and its application. The provided pigeon adenovirus strain has a deposit number of CGMCC NO.22496. The strain has strong antigenicity, can be used for the production and testing of pigeon adenovirus vaccines, and is of great significance for preventing pigeon adenovirus infection.

Description

Technical Field

[0001] The invention relates to the field of viruses, and in particular to a pigeon adenovirus SP strain and an application thereof. Technical Background

[0002] Currently, there are five genera in the Adenoviridae family, namely Mastadenovirus, Aviadenovirus, Atadenovirus, Siadenovirus and Ichtadenovirus. Adenoviruses (AdVs) can infect a variety of vertebrates, and birds are common hosts of adenoviruses. The adenovirus genome consists of a double-stranded linear [1-2] The genome lengths of different virus species in different genera vary, ranging from 26 to 45 kb. The main structural proteins of the virus are hexon protein and fiber protein, which are connected to the penton matrix in the form of non-covalent bonds to form a penton structure. Hexon protein carries antigenic determinants specific to virus species, groups, and subgroups, participates in immune responses, and is the main capsid protein of non-enveloped, icosahedral virus particles. [3-4] .

[0003] Avian adenovirus infection causes massive poultry deaths [5] , causing serious losses to the poultry industry. FAdV has a wide range of natural hosts, not only causing disease in poultry such as chickens, ducks, and geese, but also infecting poultry such as pigeons. It is a common pathogen in pigeons. [7] The first report of adenovirus infection in pigeons was in Belgium in 1984, and since then, pigeon adenovirus infection has spread worldwide. Many viruses in the genus Adenovirus can infect pigeons. In 1995, De Herdt [8] described two adenovirus-associated diseases in pigeons, termed adenovirus type I and type II infections. [8] . The naming of type I and type II has nothing to do with the difference in viral antigens, but is related to the clinical symptoms caused by adenovirus infection. Type I adenovirus (also known as classic adenovirus or type 1 pigeon adenovirus) has extremely similar clinical symptoms to young pigeon disease syndrome (YPDS). The virus mainly affects young pigeons, and sick pigeons show diarrhea, vomiting, and weight loss. Type II adenovirus (also known as type 2 pigeon adenovirus), compared to type I adenovirus, type II adenovirus affects pigeons of all ages. The typical characteristics of the disease are sudden death and extensive liver necrosis of sick pigeons. [9,10] .

[0004] Adenovirus causes serious economic losses to the pigeon industry. Therefore, isolating, identifying, and screening the FAdV strains that are prevalent in Chinese pigeon populations, cultivating vaccine strains with excellent antigenicity, and preparing corresponding vaccines will be of great significance for preventing avian adenovirus infection. Summary of the invention

[0005] To meet the needs in the above-mentioned field, the present invention provides a pigeon adenovirus strain, whose deposit number is CGMCC NO.22496.

[0006] The virus liquid containing the above-mentioned pigeon adenovirus strain also belongs to the protection scope of the present invention.

[0007] The present invention also provides a method for preparing the virus liquid, comprising: inoculating LMH cells with the pigeon adenovirus strain and culturing, harvesting cell supernatant after freezing and thawing, and obtaining the virus liquid; or inoculating SPF chickens with the pigeon adenovirus strain, harvesting liver tissue of infected dead chickens, grinding, freezing and thawing, and centrifuging to obtain the supernatant, which is the virus liquid.

[0008] The use of the above-mentioned pigeon adenovirus strain in the preparation of pigeon adenovirus vaccine also falls within the protection scope of the present invention.

[0009] The use of the above pigeon adenovirus strain in the preparation of pigeon adenovirus diagnostic reagents also falls within the protection scope of the present invention.

[0010] The virus diagnosis reagent can be an agar-amplified antigen reagent, a neutralizing antigen reagent, a HI antigen reagent or a positive serum reagent.

[0011] The use of the above-mentioned pigeon adenovirus strain in the preparation of drugs for virus therapy also falls within the protection scope of the present invention.

[0012] The drug for virus treatment can be egg yolk antibody or antiserum.

[0013] The present invention also provides a pigeon adenovirus vaccine, wherein the active component of the vaccine comprises the inactivated pigeon adenovirus strain.

[0014] The above-mentioned vaccine can be a pigeon adenovirus inactivated vaccine, a pigeon Newcastle disease-adenovirus bivalent inactivated vaccine, a pigeon Newcastle disease-adenovirus-H9 subtype avian influenza triple inactivated vaccine, a pigeon Newcastle disease-adenovirus-H9 subtype avian influenza-infectious bursal disease quadruple inactivated vaccine, a pigeon Newcastle disease-adenovirus-H9 subtype avian influenza-circovirus quadruple inactivated vaccine, or a pigeon Newcastle disease-adenovirus-H9 subtype avian influenza-rotavirus quadruple inactivated vaccine.

[0015] The present invention also provides a method for preparing the above-mentioned pigeon adenovirus, comprising: inactivating the virus liquid of the above-mentioned pigeon adenovirus strain, and then mixing and emulsifying with an adjuvant to prepare a pigeon adenovirus vaccine.

[0016] The pigeon adenovirus strain provided by the present invention is named SP strain in the laboratory. The strain can be inhibited by BUDR, is a DNA virus, is sensitive to heat, is not alkali-resistant, has no lipid capsule, cannot agglutinate the red blood cells of chickens, and is identified as serotype 4 group I avian adenovirus by genetic evolution tree analysis. The SPF chicken regression test shows that the SP strain causes 10 / 10 disease and 10 / 10 death of the test chickens, and has strong toxicity. The efficacy test results of the SP strain inactivated vaccine show that the average neutralizing antibody titer of the immunized group reached 1:97 on the 28th day after immunization, and the average neutralizing antibody titer of the control group pigeons was 0. Therefore, the SP strain of the present invention can be used as a production strain and a test strain of a pigeon adenovirus vaccine, and has a good application prospect in the prevention of avian adenovirus infection.

[0017] The pigeon adenovirus strain provided by the present invention has been patented and the deposit information is as follows:

[0018] Referenced biological material (strain): A / pigeon / Hebei / SP / 2018

[0019] Classification and nomenclature: Avian adenovirus group I serotype 4

[0020] Date of deposit: July 29, 2021

[0021] Deposit number: CGMCC No.22496

[0022] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0023] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are normal LMH cells.

[0025] Figure 2 LMH cells infected with pigeon adenovirus SP strain.

[0026] Figure 3 This is the result of PCR amplification of the DNA polymerase 52K gene fragment of the SP strain. Lane M is the DL2000 Marker and lane 1 is the amplified product.

[0027] Figure 4 This is the result of PCR amplification of the Hexon gene fragment of the SP strain DNA polymerase. Lane M is the DL2000 Marker, and lane 1 is the amplified product.

[0028] Figure 5 It is a genetic evolution tree analysis diagram of the whole genome nucleotide of the virus isolate, wherein SP18 is the SP strain of the present invention.

[0029] Figure 6 Pathological changes in SPF chickens infected with SP strain and died: pericardial effusion.

[0030] Figure 7 The pathological changes of SPF chickens infected with SP strain and died are: enlarged liver.

[0031] Figure 8 The pathological changes of SPF chickens infected with SP strain and died are: enlarged kidneys.

[0032] Fig. 9 The pathological changes of SPF chickens infected with SP strain and died are: enlarged glandular stomach.

[0033] Fig.10 The pathological changes of SPF chickens infected with SP strain and died are: bleeding in the glandular stomach mucosa. DETAILED DESCRIPTION

[0034] The present invention is described in detail below in conjunction with examples. It should be understood that the following examples are only used as explanations and illustrations of the present invention and do not limit the scope of the present invention in any way.

[0035] Biomaterials

[0036] LMH cells: chicken liver cancer cells, originated from ATCC in the United States, and were propagated, built into a cell bank, and preserved in our laboratory.

[0037] NDV Lasota strain: that is, the Lasota strain of Newcastle disease virus, provided by the Animal Immunity and Prevention Research Laboratory, Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences.

[0038] SPF chickens were purchased from Beijing Boehringer Ingelheim Weitong Biotechnology Co., Ltd. and raised in the SPF chicken house of the Animal Husbandry and Veterinary Research Institute of Beijing Academy of Agricultural and Forestry Sciences.

[0039] Pigeons were purchased from a pigeon farm in Miyun, Beijing, and raised in the animal house of the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences.

[0040] Experimental reagents

[0041] 0.25% trypsin solution was purchased from Amresco. Fetal bovine serum and DMEM culture medium were purchased from Hyclone. Column animal DNA extraction kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd. Taq Plus Master MixII was purchased from Nanjing Novozyme Biotechnology Co., Ltd. Injection white oil was purchased from ExxonMobil Corporation. S-Ben-80 and Tween-80 were purchased from Shanghai Shenyu Pharmaceutical Chemical Co., Ltd. Aluminum stearate and thimerosal aqueous solution (1%) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0042] Instruments and consumables

[0043] Biological safety cabinet, inverted microscope, CO 2 Incubator, autoclave, constant temperature water bath, 96-well microplate, single-channel and multi-channel micropipette, pipette tip, 2mL centrifuge tube, sample container, pipette, beaker, etc.

[0044] If not otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure. If not otherwise specified, the experimental methods and conditions used in the following examples are all conventional experimental methods and conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer specifications. Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0045] Example 1. Isolation, culture and identification of pigeon adenovirus SP strain

[0046] In 2018, a flock of pigeons at a pigeon farm in Hebei Province became ill and some of the pigeons died acutely. An autopsy revealed that the dead pigeons had enlarged, brittle and discolored livers, enlarged kidneys, and bleeding at the junction of the glandular stomach and gizzard.

[0047] 1. Collection and processing of diseased materials

[0048] Aseptically obtain the liver of a dead pigeon, grind it with a sterile grinder, add sterile physiological saline containing 10000 IU / mL of dual antibiotics (penicillin and streptomycin) at a volume ratio of 1:3 to prepare a virus suspension, mix well, transfer the sample to a 15mL centrifuge tube, centrifuge at 3000rpm for 5min, filter the supernatant through a 0.22μm filter for sterilization, and store it at -20℃.

[0049] 2. Newcastle disease virus test

[0050] Aseptically collect throat swabs and cloacal swabs from dead pigeons, place them in 1 mL of sterile saline containing 10,000 IU / mL of dual antibodies, vortex the swabs for 15 seconds, centrifuge at 3,000 r / min for 5 minutes, and draw 0.2 mL of the mixed supernatant of throat and cloacal swabs. Inoculate the allantoic cavity of 5 10-day-old chicken embryos, set up a negative control, seal the pinhole, and incubate at 36-37°C without turning the eggs. Within 48 hours after inoculation, illuminate the eggs once a day; thereafter, illuminate the eggs once every 4-6 hours until 120 hours; remove the chicken embryos, aseptically collect the chicken embryo allantoic fluid for blind transmission for one generation, and show that the Newcastle disease virus is negative.

[0051] 3. LMH Cell Culture

[0052] Take out LMH cell seeds from the liquid nitrogen tank for recovery. After the cell monolayer is fully grown, discard the cell culture medium, add about 1 / 10 of the volume of the cell culture medium 0.25% trypsin to rinse once, then add an appropriate amount of 0.25% trypsin for digestion. When obvious cracks appear in the cell layer, discard the trypsin solution, shake the cell bottle gently to make all the cells fall off, add DMEM culture medium containing 10% fetal bovine serum, shake the culture bottle gently to make the cells evenly dispersed, and then subculture the LMH cells at a ratio of 1:3 to 1:5 (cell number ratio).

[0053] 4. Virus Inoculation into LMH Cells

[0054] After the LMH cells have grown into a monolayer (48-72 hours), discard the cell culture medium. Take the prepared virus solution and add it to the DMEM culture medium containing 2% (v / v) fetal bovine serum at a volume ratio of 1 / 100. Then add the virus-containing DMEM culture medium to the culture flask with a monolayer of LMH cells. Incubate at 37°C and 5% CO 2 Cultivated under conditions.

[0055] 5. Observation and Harvest

[0056] Observe twice a day after infection and record the cell pathological changes. The pathological cells are round, highly refractive, evenly distributed, and have enlarged intercellular spaces. Figure 2 When the cytopathic rate reaches more than 90%, the cell fluid is harvested, the cell culture bottle is frozen, and the cell supernatant (i.e., virus fluid) is harvested after three freeze-thaw cycles, placed in a sterile container, and kept as a sample.

[0057] 6. Virulence Determination of Viruses

[0058] Prepare LMH cells, spread them in 96-well cell plates, and incubate at 37°C, 5% CO 2 The virus solution obtained after three freeze-thaw cycles was diluted 10-fold in DMEM cell culture medium, and 10 -5 , 10 -6 , 10 -7 , 10 -8 Dilute the virus solution and inoculate a 96-well cell culture plate at 0.1 mL / well. Set up 5 replicate wells for each dilution and place in a 37°C, 5% CO 2 The cells were cultured in an incubator. Seven days after inoculation, the 96-well cell culture plate was observed under a microscope, and the number of wells with and without cytopathic effects at each dilution was counted. The virus content of the tested samples was calculated using the Reed-Muench method. The results showed that the third-generation cytotoxic virus content of the isolated strain was 10 6.5 TCID 50 / 0.1ml.

[0059] 7. Plaque Purification of Virus

[0060] The virus solution obtained by the above treatment was appropriately diluted with cell culture medium, and then diluted using LMH cells according to the endpoint dilution method. The specific method is: the virus supernatant was diluted from 10 -3 ~10 -8 Serial dilutions were performed, and each dilution of the virus solution was inoculated into 1 plate of six-well plates filled with LMH cells. 0.2 ml of virus dilution solution was added to each well. The control group was replaced with cell culture medium only and placed at 37°C CO 2 Adsorb in the incubator for 1.5 h, shake once every 15 min to evenly distribute the virus, and then wash away the unadsorbed virus; mix DMEM culture medium containing 10% fetal bovine serum with an equal amount of 2% agarose (preheated), add 3 ml of the mixture to each well, wait for it to cool and solidify, and then invert and place at 37°C CO 2 After culturing in an incubator for 5 to 9 days, observe the results, pick out the diseased cells under a single plaque, and perform plaque purification twice more in the same way. The results showed that the third-generation plaque purification of the isolated strain contained ≥10 7.5 TCID 50 / 0.1ml.

[0061] 8. Virus Identification

[0062] Hemagglutination assay

[0063] 10 ml of SPF chicken whole blood was collected aseptically, and the red blood cells were repeatedly washed 3 times with sterile PBS. The red blood cells were configured to a volume percentage concentration of 1% and stored at 2-8°C. The characteristics of the virus isolate to agglutinate red blood cells were detected in a conventional manner. The NDV Lasota strain was used as a positive control and sterile PBS was used as a negative control. The results showed that the isolate could not agglutinate the red blood cells of the chicken, but NDV Lasota could agglutinate the red blood cells of the chicken.

[0064] Physical and chemical properties identification

[0065] The virus solution was treated with 5-bromo-2'-deoxyuridine (BUDR), hydrochloric acid, sodium hydroxide, chloroform, ether, etc., and an untreated positive control and a negative control of PBS solution were set. The chicken embryos were inoculated, and the results showed that BUDR could inhibit the replication of the strain. The virus is a DNA virus, sensitive to heat, not alkali-resistant, and has no lipid capsule; the virus solution treated with hydrochloric acid, ether, and chloroform can still cause lesions in chicken embryos.

[0066] PCR testing and gene sequencing

[0067] The liver of the sick pigeon was ground aseptically and sterilized saline was added to it at a volume ratio of 1:3 to prepare a virus suspension. 200 μL of the virus suspension was taken and the viral DNA was extracted using a column animal DNA extraction kit (Tiangen Biochemical). Two pairs of primers were used for identification of FAdV, and the primer sequences were as follows:

[0068] 52K-F: 5'-ATGGCKCAGATGGCYAAGG-3'

[0069] 52K-R: 5'-AGGCCCTGGGTCAAACCGA-3'

[0070] Hexon-F: 5'-CAARTTCAGRCAGACGGT-3'

[0071] Hexon-R: 5'-TAGTGATGMCGSGACATCAT-3'.

[0072] Using viral DNA as a template, PCR amplification was performed using 52K-F / 52K-R primer pair and Hexon-F / Hexon-R primer pair, respectively, according to the following reaction system and reaction procedure. After amplification, PCR products were detected by 1.5% agarose gel electrophoresis.

[0073] PCR reaction system: 2×Taq Plus Master Mix II, 25 μL; upstream primer 52K-F (10 μM), 2 μL; downstream primer 52K-R (10 μM), 2 μL; viral DNA, 5 μL; ddH 2 O, 16 μL.

[0074] PCR reaction program: 95°C for 5 min; 94°C for 50 s, 60°C for 45 s, 72°C for 30 s, 40 cycles; 70°C for 7 min; stored at 4°C.

[0075] The amplification results of the 52K-F / 52K-R primer pair are as follows Figure 3 As shown, a target fragment of 176 bp was amplified from the isolate DNA, which was consistent with the expected target fragment size.

[0076] PCR reaction system: 2×Taq Plus Master Mix II, 25 μL; upstream primer Hexon-F (10 μM), 2 μL; downstream primer Hexon-R (10 μM), 2 μL; viral DNA, 5 μL; ddH 2 O, 16 μL.

[0077] PCR reaction conditions: 95°C for 5 min; 94°C for 2 min, 60°C for 1 min, 72°C for 90 s, 35 cycles; 70°C for 2 min; stored at 4°C.

[0078] The amplification results of Hexon-F / Hexon-R primer pair are as follows Figure 4 As shown, a target fragment of 897 bp was amplified from the isolate DNA, which was consistent with the expected target fragment size.

[0079] The positive results were sequenced and analyzed for genetic evolution. According to the polymerase gene sequence comparison and genetic evolution tree analysis, the results showed that the isolate was most similar to the genus I fowl adenovirus type 4 ( Figure 5 ). Therefore, the virus isolate was identified as serotype 4 group I fowl adenovirus and named SP strain. The strain has been patented and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.22496, and the classification name is fowl adenovirus group I serotype 4, and the deposit date is July 29, 2021.

[0080] Example 2. Animal regression test of pigeon adenovirus SP strain

[0081] Twenty 70-day-old SPF chickens were used, 10 of which were used as the challenge group and 10 as the negative control group. Each chicken in the challenge group was injected intramuscularly with 10 6.0 TCID 50 0.1ml of SP strain virus liquid was injected into the negative control group, and no virus liquid was injected into the negative control group. After 7 days of continuous observation, all chickens were autopsied and the incidence and mortality of the chickens were recorded. The results are shown in Table 1. The SP strain caused 10 / 10 diseases and 10 / 10 deaths in the chickens in the challenge group. The chickens in the challenge group began to become ill within 24 hours, with depression and green loose stools. The clinical symptoms worsened after 48 hours, with feathers standing upright, less food intake, curling up in a corner, crouching and lethargy, and began to die. Autopsy showed yellow pericardial effusion, enlarged liver, sometimes with purple-red spots; enlarged kidneys, and bleeding spots at the junction of the gizzard and glandular stomach, such as Figure 6-10 There were no clinical manifestations or autopsy changes in the negative control group.

[0082] Table 1 Results of the challenge test of the SP strain of the present invention on 70-day-old SPF chickens

[0083]

[0084] Example 3. Preparation of pigeon adenovirus vaccine

[0085] 1. Preparation of antigens for vaccine production

[0086] (1) Preparation of LMH cells

[0087] Take out the LMH cell seeds from the liquid nitrogen tank for recovery. After the cells are fully grown in a monolayer, discard the cell culture medium, add 0.25% trypsin solution with a volume of 1 / 10 of the cell culture medium to rinse once, then add an appropriate amount of 0.25% trypsin to digest, and discard the trypsin solution when cracks appear in the cell layer. Gently shake the cell bottle to make all the cells fall off, add DMEM culture solution containing 10% (v / v) fetal bovine serum, gently blow the cell solution to make the cells evenly dispersed, and then subculture the LMH cells at a ratio of 1:3 to 1:5 (cell number ratio).

[0088] (2) Inoculation and culture

[0089] After the cells have grown to a monolayer (48-72 hours), the cell culture medium is discarded. The virus solution (SP strain) prepared in Example 1 is added to a DMEM culture medium containing 2% (v / v) fetal bovine serum at a volume ratio of 1 / 100, and then the virus-containing DMEM culture medium is added to a culture flask filled with LMH cells and incubated at 37°C, 5% CO 2 Cultivated under conditions.

[0090] (3) Observation and Gains

[0091] After infection, the animals were observed twice a day and the cytopathic effects were recorded. The cytopathic cells were round, highly refractive, evenly distributed, and had enlarged intercellular spaces ( Figure 2 ). When the cytopathic rate reaches more than 90%, the cell fluid is harvested, the cell culture bottle is frozen, and after three freeze-thaw cycles, it is placed in a sterile container, and a sample is retained for testing.

[0092] (4) Inspection

[0093] The virus solution obtained after three freeze-thaw cycles was subjected to sterility testing and virus content determination according to the current appendix of the Chinese Veterinary Pharmacopoeia. The results showed that no bacteria grew and the virus content in each 0.1 mL of virus solution was ≥10 7.0 TCID 50 .

[0094] (5) Virus liquid inactivation

[0095] Add 10% formaldehyde solution to the virus solution that has passed the test, so that the final concentration of the formaldehyde solution reaches 0.2% by volume, stir while adding, mix the formaldehyde solution and the virus solution thoroughly, and inactivate at 37°C, starting from the time when the temperature of the antigen solution reaches 37°C, and inactivate for 16 hours. After the inactivation is completed, take a sample for inactivation test, and store the inactivated virus solution at 2-8°C for no more than 30 days.

[0096] (6) Semi-finished product inspection

[0097] Sterility test: According to the appendix of the current "Chinese Veterinary Pharmacopoeia", the inactivated virus liquid was subjected to a sterility test, and the results showed no bacterial growth.

[0098] Virus content determination: The virus solution before inactivation was diluted 10 times in DMEM medium, and 10 -6 , 10 -7 , 10 -8 Three dilutions were inoculated into 5 wells of monolayer LMH cells, 0.1 mL per well, and placed at 37°C and 5% CO 2 The cells were cultured under the same conditions and observed for 192 h. TCID 50 The results showed that the virus content in each 0.1mL virus liquid was ≥10 7.0 TCID 50 .

[0099] Inactivation test: The inactivated virus solution was diluted 10 times and inoculated into 4 T25 cell culture flasks that were already full of monolayer LMH cells. At the same time, a flask of cells that were not inoculated with virus was set as a control. The cells were incubated at 37°C and 5% CO. 2 The cells were cultured under the same conditions and observed for 192 hours. No cytopathic effect was observed in the sample culture bottle or the cell control bottle. The cell cultures without cytopathic effect at 192 hours were collected, frozen and thawed three times, and then blindly propagated for another generation. The cells were cultured for another 192 hours. No cytopathic effect was observed in the sample bottle, which was considered to be completely inactivated.

[0100] 2. Preparation of Oil-Adjuvanted Inactivated Vaccine

[0101] Preparation of oil phase: Take 94 parts by weight of white oil for injection, add 2% (by weight) aluminum stearate, and heat while stirring until it becomes completely transparent. Then add 6 parts by weight of Siben-80, mix well, and sterilize under high pressure to obtain the oil phase.

[0102] Preparation of aqueous phase: Take 96 parts by weight of the inactivated virus solution, add 4 parts by weight of sterilized Tween-80, and shake thoroughly until the Tween-80 is completely dissolved to obtain an aqueous phase.

[0103] Emulsification: Pour 2 parts by weight of the oil phase into an emulsification tank, slowly stir and slowly add 1 part by weight of the water phase, mix at a medium speed and then emulsify at a high speed, i.e., 2900 rpm for 40 to 60 minutes. Before the end of emulsification, add a 1% by volume aqueous solution of thimerosal to a final concentration of 0.01% by volume. After emulsification, take 10 mL and add it to a centrifuge tube, centrifuge at 3500 r / min for 15 minutes, and if no stratification occurs, the pigeon adenovirus vaccine is obtained.

[0104] Packaging: Pack in quantitative quantities, seal with a lid, label, and store at 2-8℃.

[0105] Example 4. Testing of pigeon adenovirus vaccine

[0106] The pigeon adenovirus vaccine prepared in Example 3 was subjected to the following tests.

[0107] 1. Vaccine properties

[0108] Appearance: White homogeneous emulsion.

[0109] Dosage form: Oil-in-water type. Take a clean pipette, draw a small amount of vaccine and drop it into cold water. Except for the first drop, it should not spread.

[0110] Stability: Pipette 10 mL of vaccine, add into a centrifuge tube, centrifuge at 3500 r / min for 15 min, and the aqueous phase precipitated at the bottom of the tube should not exceed 0.5 mL.

[0111] Viscosity: The viscosity test was carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia" and met the requirements.

[0112] 2. Carry out filling quantity inspection according to the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", and the filling quantity complies with the regulations.

[0113] 3. Sterility test was carried out according to the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", and the results showed no bacterial growth.

[0114] 4. Safety inspection

[0115] Fifteen 5-week-old SPF chickens were used, 10 of which were used as the immunization group and 5 as the control group. Each chicken in the immunization group was injected with 1.0 mL of the pigeon adenovirus vaccine prepared in Example 3 into the chest muscle. The control group was not injected with the vaccine. The chickens in the immunization group and the control group were raised under the same conditions and observed for 14 consecutive days, and the feeding, drinking and clinical conditions of the experimental chickens were recorded. The results showed that the immunization group chickens ate and drank normally, and no local or systemic adverse reactions occurred. The autopsy of the immunization group chickens found that the vaccine absorption effect was good.

[0116] Fifteen 60-day-old non-immune pigeons were used, 10 of which were used as the immunization group and 5 as the control group. Each pigeon in the immunization group was injected with 0.4 mL of the pigeon adenovirus vaccine prepared in Example 3 into the chest muscle. The control group was not injected with the vaccine. The pigeons in the immunization group and the control group were raised under the same conditions and observed for 14 consecutive days, and the feeding, drinking and clinical conditions of the experimental pigeons were recorded. The results showed that the immunization group pigeons ate and drank normally, and no local and systemic adverse reactions occurred. The autopsy of the immunization group pigeons found that the vaccine absorption effect was good.

[0117] 5. Effectiveness test

[0118] The efficacy of the vaccine was tested using serological methods and immune challenge methods.

[0119] (1) Serological methods to test vaccine efficacy

[0120] Fifteen 5-week-old SPF chickens were used, 10 of which were used as the immunization group and 5 as the control group. Each chicken in the immunization group was injected with 0.3 mL of the pigeon adenovirus vaccine prepared in Example 3 into the chest muscle. The chickens in the control group were not vaccinated. On the 28th day after immunization, blood was collected from the wing vein of the test chickens to separate serum and measure adenovirus neutralizing antibodies. The average neutralizing antibody titer of the control group chickens should not be higher than 1:2, and the average neutralizing antibody titer of the immunization group chickens should not be lower than 1:16.

[0121] Fifteen 60-day-old non-immune pigeons were used, 10 of which were used as the immunization group and 5 as the control group. Each pigeon in the immunization group was injected with 0.2 mL of the pigeon adenovirus vaccine prepared in Example 3 into the chest muscle. The control group was not injected with the vaccine. On the 28th day after immunization, blood was collected from the wing vein of the test pigeons to separate serum and measure adenovirus neutralizing antibodies. The average neutralizing antibody titer of the control group pigeons should not be higher than 1:2, and the average neutralizing antibody titer of the immunized group pigeons should not be lower than 1:16.

[0122] The neutralizing antibody assay is as follows:

[0123] The neutralization test antigen was the virus solution (SP strain) prepared in Example 1, with a virus content of ≥10 6.0 TCID 50 / 0.1mL; the positive serum was prepared by immunizing SPF chickens with SP strain in our laboratory, taking the immune chicken blood and separating the serum, and the neutralizing antibody titer has been determined; the negative serum was collected from SPF chicken blood and prepared by separating the serum, and the neutralizing antibody titer is not higher than 1:2.

[0124] Before the test, the serum to be tested was inactivated in a 56°C water bath for 30 min. LMH cells were cultured according to conventional methods. 0.10 mL of LMH cell solution (15,000 / well) was added to each well of a 96-well cell culture plate and placed at 37°C and 5% CO. 2 Culture in an incubator for 24 hours. After the cells grow into a monolayer, add DMEM culture medium to the 96-well cell plate (first plate), add 0.05 mL to each well, take 0.05 mL of inactivated serum to be tested and add it to the first well, mix thoroughly and take 0.05 mL and add it to the second well, and perform 2-fold serial dilutions in sequence until 1:2 24 After the serum is diluted, it is diluted from 1:2 24 to 1:2 1 Add 0.05 mL of virus suspension (titer 200 TCID 50 / 0.025mL), shake well to mix, and place at 37°C, 5% CO 2 Incubate for 1 hour. Discard the supernatant from the culture plate (the second set of plates) covered with LMH cell monolayers, add 0.15 mL of DMEM culture medium containing 2% fetal bovine serum to each well; 1 to 1:224 Transfer 0.05 mL of virus neutralizing solution per well of the first plate to the corresponding wells of the second plate, using 1 pipette tip for each sample. Shake the cell culture plate for 30 s. During the test, set up normal LMH cell control, negative serum control, positive serum control, and virus control. Place the second plate in an incubator at 37 °C and 5% CO 2 2 incubator for culturing, and observe for 8 days. When all the virus control wells show lesions, the normal LMH cell control presents a good monolayer, the positive serum neutralizing antibody titer is within the known range, and the negative serum neutralizing antibody titer is not higher than 1:2, the test results are valid. The highest dilution concentration of the serum that completely inhibits the appearance of cell lesions is the neutralizing antibody titer of this serum.

[0125] The results showed that the average neutralizing antibody titer of the immunized chickens reached 1:1024 on the 28th day after immunization, while that of the control group chickens was 0. The average neutralizing antibody titer of the immunized pigeons reached 1:97 on the 28th day after immunization, while that of the control group pigeons was 0.

[0126] (2) Testing the vaccine efficacy by the immunization and challenge method

[0127] Use 20 5-week-old SPF chickens, with 10 as the immunized group and 10 as the control group. Inject 0.3 mL of the pigeon adenovirus vaccine prepared in Example 3 into the chest muscle of each chicken in the immunized group. The control group is not injected with the vaccine. On the 28th day after immunization, all the test chickens are challenged with the pigeon adenovirus SP strain of the present invention, and 0.1 ml of the diluted SP strain virus solution (virus content 10 6.0 TCID 50 ) is injected into the chest muscle of each chicken, and observe for 7 days. At least 9 chickens in the control group should show disease (showing listlessness, green feces, etc., and severe cases may die; autopsy shows lesions such as pericardial effusion, enlarged and necrotic liver, etc.), and at least 9 chickens in the immunized group should be protected (without clinical symptoms and autopsy lesions).

[0128] The results showed that after challenge with the SP strain, 10 / 10 chickens in the control group showed disease, while 10 / 10 chickens in the immunized group were protected, and the protection rate was 100%.

[0129] Table 2 Results of the challenge protection test of the pigeon adenovirus SP strain of the present invention

[0130]

[0131] 6. Determine the residual amounts of formaldehyde and mercury-based preservatives in the vaccine according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and the results meet the requirements of the General Principles for Veterinary Biological Products.

[0132] References:

[0133] [1] HARRACH B.Molecular evolution of adenoviruses[J].CurrentTopics in Microbiology and Immunology,2003,272:3-35.

[0134] [2]NICZYPORUK JS, KOZDRUN W, CZEKAJ H, et al. Detection of fowladenovirus D strains in wild birds in Poland by loop mediated isothermalamplification(LAMP)[J]. BMC Veterinary Research, 2020, 16(1):1-12.

[0135] [3]VEREECKEN M,DE HERDT P,DUCATELLA R.Adenovirus infections inpigeons:A review[J].Avian Pathology,1998,27(4):333-338.

[0136] [4] Hu Qinghai, Huang Jianfang. Pigeon adenovirus infection[J]. China Poultry, 1999, 21(3): 41-42.

[0137] [5]ZHAO J, ZHONG Q, ZHAO Y, et al. Pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated within inclusion body hepatitis and hydropericardium syndrome in China[J]. PLoS One, 2015, 10(7):e133073.

[0138] [6] Yuan Wanzhe, Li Yubao, Wang Jianchang, et al. Preliminary study on pericardial effusion-hepatitis syndrome in chickens[J]. Chinese Journal of Veterinary Science, 2016(2):157-160.

[0139] [7] SALF Y M. Avian Diseases[M]. Translated by Gao Jingliang, Gao Fu, Suo Xun. 11th edition. Beijing: China Agriculture Press, 2005: 376-384.

[0140] [8] De Herdt P, Ducatelle R, Lepoudre C, et al. An epidemic of fatalhepatic necrosis of viral origin in racing pigeons (Columba livia) [J]. AvianPathology, 1995, 24(3): 475-483.

[0141] [9] Bao Xiangjia, Han Xianglin, Sun Junhua, et al. Proceedings of the 9th Representative Conference of the Poultry Branch of the Chinese Society of Animal Husbandry and Veterinary Medicine and the 16th National Poultry Academic Symposium [C]. Yangzhou: China Poultry, 2013: 2.

[0142]

[10] Jian Xiuyan. Diagnosis and treatment of a pigeon adenovirus case[J]. Breeding Technology Consultant, 2012(6):206.

[0143]

[11] A,MAREK A,GRAFL B,et al.Real-time PCR assay for universal detection and quantitation of all five species of fowl adenoviruses(FAdV-A toFAdV-E)[J].Journal of Virological Methods, 2012,183(2):147-153.

[0144]

[12] MEULEMANS G, BOSCHMANS M, Van D, et al. Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses[J]. Avian Pathology Journal of the Wvpa, 2001, 30(6): 655-660.

Claims

1. Pigeon adenovirus strain, its deposit number is CGMCC NO.22496.

2. A virus solution comprising the pigeon adenovirus strain according to claim 1.

3. The method for preparing the virus solution according to claim 2, It is characterized in that include: Inoculate LMH cells with the pigeon adenovirus strain and culture them, and harvest the cell supernatant after freezing and thawing to obtain the virus solution; Alternatively, the pigeon adenovirus strain is used to inoculate SPF chickens, and the liver tissue of the infected dead chickens is harvested, ground, frozen and thawed, and centrifuged to obtain the supernatant, which is the virus liquid.

4. Use of the pigeon adenovirus strain according to claim 1 in preparing a pigeon adenovirus vaccine.

5. Use of the pigeon adenovirus strain according to claim 1 in preparing a reagent for pigeon adenovirus diagnosis.

6. Use of the pigeon adenovirus strain according to claim 1 in preparing drugs for virus therapy.

7. The use according to claim 6, It is characterized in that The drug for virus treatment is egg yolk antibody or antiserum.

8. A pigeon adenovirus vaccine, It is characterized in that The active ingredient of the vaccine comprises the inactivated pigeon adenovirus strain of claim 1.

9. The pigeon adenovirus vaccine according to claim 8, It is characterized in that The vaccine is a pigeon adenovirus inactivated vaccine, a pigeon Newcastle disease-adenovirus bivalent inactivated vaccine, a pigeon Newcastle disease-adenovirus-H9 subtype avian influenza triple inactivated vaccine, a pigeon Newcastle disease-adenovirus-H9 subtype avian influenza-infectious bursal disease quadruple inactivated vaccine, a pigeon Newcastle disease-adenovirus-H9 subtype avian influenza-circovirus quadruple inactivated vaccine, or a pigeon Newcastle disease-adenovirus-H9 subtype avian influenza-rotavirus quadruple inactivated vaccine.

10. The method for preparing the pigeon adenovirus vaccine according to claim 8, It is characterized in that include: The virus solution described in claim 2 is inactivated, and then mixed and emulsified with an adjuvant to prepare a pigeon adenovirus vaccine.

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