H5 and H7 subtype avian influenza virus homeomorphic proliferation culture method

Abstract

The invention provides a culture method for homeomorphic proliferation of H5 and H7 subtype avian influenza viruses, which adopts a mode of mixing two strains and then carrying out homeomorphic inoculation, overcomes the technical defects of tedious operation, long time and the like during proliferation of various virus antigens in a traditional preparation method, greatly improves the efficiency of antigen production and saves the production cost. The proliferation of two viruses is realized in one chick embryo, the immune stress can also be reduced, and the antigen prepared by adopting the preparation method is good in safety and good in immune effect.

Description

Technical Field

[0001] The invention belongs to the field of veterinary biopharmaceuticals, and in particular relates to a culture method for isoembryonic proliferation of H5 and H7 subtype avian influenza viruses. Background Art

[0002] Avian influenza (AI) is a highly contagious disease caused by influenza A virus that infects poultry and wild birds. Due to the different antigenicity of hemagglutinin (HA) and neuraminidase (NA) of different avian influenza viruses (AIV), 16 specific HAs and 9 specific NAs have been found in poultry, named H1~H16, N1~N9 respectively. Different HAs and different NAs can form more than 100 subtypes of avian influenza viruses. According to the pathogenicity of avian influenza viruses, AIV can be divided into high pathogenic avian influenza viruses (High Pathogenic Avian Influenza Virus, HPAVI) and low pathogenic avian influenza viruses (Low Pathogenic Avian Influenza Virus, LPAIV). At present, the H5 or H7 subtype avian influenza viruses prevalent in China are all HPAIV.

[0003] For the current production of H5 and H7 subtype avian influenza virus antigens, the traditional method can only use each virus to be inoculated and cultured separately, and then the cultured viruses are mixed to prepare the water phase, and then mixed with the oil phase in a certain proportion to prepare the oil emulsion inactivated vaccine. Since the yield of each virus antigen is affected by the chicken embryo culture conditions and the harvest volume, the production cost is high, and the time required to prepare multiple virus antigens is long, and the process cycle is long, which has a certain impact on the product yield. Summary of the invention

[0004] The purpose of the present invention is to provide a method for culturing the homozygous proliferation of H5 and H7 subtype avian influenza viruses.

[0005] The present invention also aims to reduce vaccine production costs and immune stress through homozygous vaccination.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] A method for culturing H5 and H7 subtype avian influenza viruses for homozygous proliferation, the method comprising the following steps:

[0008] (1) Select 11-day-old chicken embryos and inoculate the allantoic cavity with H5 or H7 subtype avian influenza viruses;

[0009] (2) Incubate for 72 hours and discard any chicken embryos that die within 72 hours;

[0010] (3) Take out all the chicken embryos, place them upright with the air chamber facing upward, and cool them at 2 to 8°C for 4 to 24 hours;

[0011] (4) taking out the cooled chicken embryo and absorbing the chicken embryo allantoic fluid;

[0012] (5) Store the harvested chicken embryo allantoic fluid at 4°C.

[0013] Furthermore, the H5 subtype avian influenza virus is the H5N2 subtype avian influenza virus rGD59 strain or the H5N2 subtype avian influenza virus rHN5801 strain.

[0014] Furthermore, the H7 subtype avian influenza virus is the H7N9 subtype avian influenza virus rHN7903 strain.

[0015] Furthermore, the method of inoculating H5 and H7 subtype avian influenza viruses in step (1) is to dilute the H5N2 subtype avian influenza virus rGD59 strain into 10 5.5 EID 50 , H7N9 subtype avian influenza virus rHN7903 strain was diluted to 10 4.5 EID 50 , then mix in a 1:1 ratio.

[0016] Furthermore, the method of inoculating H5 and H7 subtype avian influenza viruses in step (1) is to dilute the H5N2 subtype avian influenza virus rHN5801 strain into 10 5.5 EID 50 , H7N9 subtype avian influenza virus rHN7903 strain was diluted to 10 3.5 EID 50 , and then mix in a 1:1 ratio.

[0017] The invention discloses an application of the culture method for isoembryonic proliferation of H5 and H7 subtype avian influenza viruses in the preparation of avian influenza vaccines.

[0018] By adopting the above technical solution, the present invention has the following beneficial effects:

[0019] The present invention overcomes the technical defects of the traditional preparation method that when multiple viral antigens are propagated, it is difficult to propagate them in the same embryo, the operation is cumbersome, and the time is long, and the efficiency of antigen production is greatly improved and the production cost is saved. The propagation of two viruses in one chicken embryo can also reduce immune stress. The antigens prepared by the preparation method have good safety and good immune effect. DETAILED DESCRIPTION

[0020] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention.

[0021] Example 1: Culture method for homozygous proliferation of H5 and H7 subtype avian influenza viruses

[0022] 1. Cultivation methods and steps

[0023] The H5N2 subtype avian influenza virus rGD59 strain, the H5N2 subtype avian influenza virus rHN5801 strain, and the H7N9 subtype avian influenza virus rHN7903 strain were isolated and extracted by the laboratory of South China Agricultural University.

[0024] (1) Dilute the H5N2 subtype avian influenza virus rGD59 strain to 10 4.5 EID 50 , 10 5.5 EID 50 , 10 6.5 EID 50 There were three groups in total. The H7N9 subtype avian influenza virus rHN7903 strain was diluted to 10 3.5 EID 50 , 10 4.5 EID 50 , 10 5.5 EID 50 There are three groups in total. After dilution, the two virus liquids are mixed in a ratio of 1:1 to form combination 1, combination 2 and combination 3.

[0025] (2) Dilute the H5N2 subtype avian influenza virus rHN5801 strain to 10 4.5 EID 50 , 10 5.5 EID 50 , 10 6.5 EID 50 There were three groups in total. The H7N9 subtype avian influenza virus rHN7903 strain was diluted to 10 3.5 EID 50 , 10 4.5 EID 50 , 10 5.5 EID 50 There are three groups in total. After dilution, the two virus solutions are mixed in a ratio of 1:1 to form combination 4, combination 5 and combination 6.

[0026] (3) Different combinations of virus mixtures were inoculated into 11-day-old non-immune susceptible chicken embryos through the allantoic cavity at 0.1 ml per embryo. The pinholes were sealed and the eggs were incubated at 36°C.

[0027] (4) Within 48 hours after inoculation, the eggs should be candled once a day, and dead embryos within 48 hours should be discarded. Thereafter, the eggs should be candled once every 4 to 6 hours, and dead embryos should be removed at any time until 72 hours, when all the embryos, dead or not, should be removed, with the air chambers upright, and cooled at 2 to 8°C for 4 to 24 hours.

[0028] (5) Take out the cooled chicken embryo, disinfect the eggshell at the air cell, aseptically peel off the eggshell at the air cell, remove the eggshell membrane, cut the chorioallantoic membrane and amnion (be careful to prevent the yolk from rupturing), and absorb the embryonic fluid. Each chicken embryo should be carefully inspected before absorbing the embryonic fluid. Any embryo that is corrupt, has turbid embryonic fluid, or has any suspected contamination should be discarded. The harvested chicken embryonic allantoic fluid should be stored at 4°C for later use.

[0029] 2. Determination of virus titer: RNA was extracted from the homoembryonic antigen liquid obtained by inoculation at different dilution multiples using a nucleic acid extraction kit, and the virus titer was determined by fluorescent quantitative PCR to determine the optimal virus dilution multiple.

[0030] Table 1 Effect of different dilution multiples of homozygous inoculation of rGD59 / rHN7903 on virus titer

[0031]

[0032] As shown in the data in Table 1, the H5 subtype avian influenza virus rGD59 strain was diluted to 10 5.5 EID 50 The H7 subtype avian influenza virus rHN7903 strain was diluted to 10 4.5 EID 50 , inoculated into 11-day-old non-immune susceptible chicken embryos and cultured at 36°C for 72 hours, the virus titer of the rGD59 strain in the obtained virus solution can reach 10 8.5 EID 50 , greater than 10 8.0 EID 50 , and is greater than the virus titer obtained at other dilution multiples; the virus titer of rHN7903 strain in the virus solution can reach 10 8.2 EID 50 , greater than 10 8.0 EID 50 , and the virus titer was greater than that obtained by other dilution multiples. Therefore, the H5 subtype avian influenza virus rGD59 strain was diluted to 10 5.5 EID 50 The H7 subtype avian influenza virus rHN7903 strain was diluted to 104.5 EID 50 , mixing the two virus solutions in a 1:1 ratio and inoculating them into chicken embryos can obtain the homoembryo virus solution with the best virus titer.

[0033] Table 2 Effect of different dilution multiples of homozygous inoculation of rHN5801 / rHN7903 on virus titer

[0034]

[0035] As shown in Table 2, the H5 subtype avian influenza virus rHN5801 strain was diluted to 10 5.5 EID 50 The H7 subtype avian influenza virus rHN7903 strain was diluted to 10 3.5 EID 50 , inoculated into 11-day-old non-immune susceptible chicken embryos and cultured at 36°C for 72 hours, the virus titer of the rHN5801 strain in the obtained virus solution can reach 10 8.5 EID 50 , greater than 10 8.0 EID 50 , and is greater than the virus titer obtained at other dilution multiples; the virus titer of rHN7903 strain in the virus solution can reach 10 8.4 EID 50 , greater than 10 8.0 EID 50 , and the virus titer was greater than that obtained by other dilution multiples. Therefore, the H5 subtype avian influenza virus rHN5801 strain was diluted to 10 5.5 EID 50 The H7 subtype avian influenza virus rHN7903 strain was diluted to 10 3.5 EID 50 , mixing the two virus solutions in a 1:1 ratio and inoculating them into chicken embryos can obtain the homoembryo virus solution with the best virus titer.

[0036] Example 2: Evaluation of the immune effect of H5 and H7 subtype avian influenza virus homozygous proliferation antigens

[0037] 1. Test methods and steps

[0038] (1) Concentration of virus solution: The harvested rGD59 / rHN7903 isoenzyme virus solution and rHN5801 / rHN7903 isoenzyme virus solution were concentrated to 1 / 2 of the original volume using ultrafiltration concentration equipment.

[0039] (2) Inactivation of virus solution: Add formaldehyde solution to the rGD59 / rHN7903 isoembryo virus solution and rHN5801 / rHN7903 isoembryo virus solution that have passed the test to a final concentration of 0.1%, and inactivate them at 37°C for 24 hours.

[0040] (3) Preparation of inactivated vaccines

[0041] Preparation of oil phase: Take 94 parts (by weight) of white oil for injection, add 6 parts (by weight) of Siben-80, mix thoroughly, sterilize and set aside.

[0042] Preparation of the aqueous phase: Mix 48.5 parts (by weight) of inactivated rGD59 / rHN7903 homoembryonic antigen solution (2×) and 48.5 parts (by weight) of rHN5801 / rHN7903 homoembryonic antigen solution (2×), add 3 parts (by weight) of sterilized Tween-80, and stir thoroughly until the Tween-80 is completely dissolved.

[0043] Emulsification: The oil emulsion inactivated vaccine was prepared by using laboratory emulsification equipment with the water phase and oil phase in a ratio of 1:2 (volume ratio), and was named Vaccine 1. A recombinant avian influenza virus (H5+H7) trivalent inactivated vaccine (H5N2 rHN5801 strain + rGD59 strain, H7N9 rHN7903 strain) was taken as a control vaccine and was named Vaccine 2.

[0044] (4) Vaccine immune efficacy testing

[0045] Twenty 21-day-old SPF chickens were randomly divided into two groups, A and B, with 10 chickens / group. Vaccine 1 and vaccine 2 were subcutaneously inoculated through the neck, 0.3 ml / chicken, and 5 SPF chickens of the same age were not immunized as controls. Blood was collected from each group 21 days after immunization to separate serum, and the HI antibody titer in the serum was determined using hemagglutination inhibition test antigens of rGD59 strain of H5N2 subtype recombinant avian influenza virus and rHN5801 strain, and hemagglutination inhibition test antigens of rHN7903 strain of H7N9 subtype recombinant avian influenza virus.

[0046] 2. Test results

[0047] As shown in the data of Table 3, 21 days after immunization of 3-week-old SPF chickens with the recombinant avian influenza virus (H5+H7) trivalent inactivated vaccine (vaccine 1) prepared with rGD59 / rHN7903 homoembryonic antigens and rHN5801 / rHN7903 homoembryonic antigens, the average titers of HI antibodies against rHN5801, rGD59 and rHN7903 in the serum were 8.7㏒ 2 、8.8㏒ 2 and 8.0㏒ 2The HI antibody titers of rHN5801, rGD59 and rHN7903 strains in the serum of the recombinant avian influenza virus (H5+H7) trivalent inactivated vaccine (vaccine 2) prepared with single embryonic antigen liquid were basically equivalent. This proves that the immune effect of the vaccine prepared with the same embryonic antigen is not affected.

[0048] Table 3 Results of immune efficacy of different vaccines on SPF chickens

[0049]

[0050]

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for culturing H5 and H7 subtype avian influenza viruses for homozygous proliferation, characterized in that: The culture method comprises the following steps: (1) Select 11-day-old chicken embryos and inoculate the allantoic cavity with H5 or H7 subtype avian influenza viruses; (2) Incubate for 72 hours and discard any chicken embryos that die within 72 hours; (3) Take out all the chicken embryos, place them upright with the air chamber facing upward, and cool them at 2 to 8°C for 4 to 24 hours; (4) taking out the cooled chicken embryo and absorbing the chicken embryo allantoic fluid; (5) Store the harvested chicken embryo allantoic fluid at 4°C.

2. The method for culturing the homozygous proliferation of H5 and H7 subtype avian influenza viruses according to claim 1, characterized in that: The H5 subtype avian influenza virus is an H5N2 subtype avian influenza virus rGD59 strain or an H5N2 subtype avian influenza virus rHN5801 strain.

3. The method for culturing the homozygous proliferation of H5 and H7 subtype avian influenza viruses according to claim 1, characterized in that: The H7 subtype avian influenza virus is the H7N9 subtype avian influenza virus rHN7903 strain.

4. The method for culturing the homozygous proliferation of H5 and H7 subtype avian influenza viruses according to claim 1, characterized in that: The method of inoculating H5 and H7 subtype avian influenza viruses in step (1) is to dilute the rGD59 strain of H5N2 subtype avian influenza virus into 10 5.5 EID 50 , H7N9 subtype avian influenza virus rHN7903 strain was diluted to 10 4.5 EID 50 , and then mix in a 1:1 ratio.

5. The method for culturing the homozygous proliferation of H5 and H7 subtype avian influenza viruses according to claim 1, characterized in that: The method of inoculating H5 and H7 subtype avian influenza viruses in step (1) is to dilute the H5N2 subtype avian influenza virus rHN5801 strain into 10 5.5 EID 50 , H7N9 subtype avian influenza virus rHN7903 strain was diluted to 10 3.5 EID 50 , and then mix in a 1:1 ratio.

6. Use of the method for culturing the homozygous proliferation of H5 and H7 subtype avian influenza viruses as claimed in claim 1 in the preparation of avian influenza vaccines.

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