H6 subtype avian influenza virus strain and application thereof in preparation of inactivated vaccine

Abstract

The application relates to the technical field of biological products, and discloses an H6 subtype avian influenza virus strain and application thereof in vaccines. The applicant has isolated an H6 subtype avian influenza virus strain with high immunogenicity from clinical samples in Hebei, the egg production of chickens infected with the virus is reduced by 30%, and more than 10% of the chickens are injured or dead, the preservation number of the strain is CCTCC NO: V202657. The strain has a high proliferation speed and high yield on chicken embryos, is suitable for large-scale factory vaccine production, and the inactivated vaccine prepared from the strain can induce high-level hemagglutination inhibition antibodies after inoculation on poultry, can provide 100% immune protection against attacks of homologous strains, significantly inhibits clinical symptoms and virus discharge, and has extremely high clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to an H6 subtype avian influenza virus strain and its application in vaccines. Background Technology

[0002] Avian influenza (AI) is a highly contagious disease of birds caused by type A influenza viruses. In recent years, the detection rate of strains such as H6N1, H6N2, and H6N6 has been increasing year by year in major poultry producing areas in Hebei Province and surrounding provinces of my country.

[0003] Although H6 is usually low pathogenicity (LPAI), its harm to the egg-laying hen industry is obvious. As an immunosuppressive and chronic wasting disease, infected flocks exhibit severe drops in egg production (15-40%) and respiratory symptoms, causing significant direct economic losses to farmers.

[0004] The H6 subtype virus is referred to by the academic community as a "reservoir" or "exchange platform" for the avian influenza genome. Li et al. (2017) found through continuous recombination analysis of circulating strains in southern and northern China that the internal genes of the H6 virus exhibit extremely complex recombination patterns. In particular, the widely prevalent H9N2 subtype avian influenza virus provides a large amount of internal gene matrix (such as PB2, PA, M, and NS genes) for the H6 virus. This internal gene scaffold contributed by H9N2 greatly enhances the adaptability and replication level of the H6 virus in poultry, enabling the H6 strain to achieve rapid antigenic shift and virulence evolution in the wild environment, increasing the difficulty of clinical prevention and control. In addition, the pathogenicity of the H6 virus is not limited to the respiratory and reproductive systems; aerosol exposure can also significantly enhance the infectivity of LPAI H6N1 virus in chicken flocks. The virus can rapidly replicate in the trachea, lungs, and intestines, and induce a surge in inflammatory factors such as interferon (IFN-γ) and interleukins (IL-6, IL-1β). This highly efficient respiratory transmission capability makes the H6 virus extremely prone to explosive outbreaks in closed, standardized farms.

[0005] Faced with the increasingly severe epidemic trend of H6 subtype avian influenza virus, the current prevention and control strategy mainly relies on biosafety measures and vaccination. However, due to the significant antigenic differences between the H6 virus and these subtypes, existing commercial vaccines cannot provide effective protection. Currently, there is a severe shortage of standard vaccines specifically targeting the currently circulating H6 subtype. Selecting a dominant strain with broad-spectrum immunogenicity, high titer, and the ability to provide complete protection against the current epidemic areas such as Hebei is a critical issue that urgently needs to be addressed in the current prevention and control work. These emerging epidemic pathogens place high demands on vaccine strain selection. Taking the common commercial vaccine strain WD strain of H9 subtype influenza virus as an example, the study "Observation on the Protective Effect of H9 Subtype Avian Influenza Virus WD Strain Inactivated Vaccine against H9 Subtype Avian Influenza Epidemic Strains" conducted by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences, pointed out that the hemagglutination titer of influenza isolates was generally between 8 and 9, with only one strain reaching 10, and only one strain achieving a viral titer of 10. 8.1 / 0.1ml, the rest were all in the range of 10 7 / 0.1ml-10 8 The dosage is between 0.1 ml and 10 ml. Furthermore, referring to the influenza vaccine quality standards in the *Chinese Veterinary Pharmacopoeia*, the following criteria must be met to qualify as a candidate vaccine strain: 1. The isolate must be able to be stably passaged in chicken embryos after rapid adaptation, and must be free from exogenous microbial contamination, meeting the requirements for establishing a seed bank; 2. The hemagglutination titer of the progeny influenza virus must be >2^9, and simultaneously meet the EID (Enhanced Individual's Difference) requirement. 50 ≥ 10 8.0 / 0.1ml is required to meet the needs of vaccine preparation; otherwise, an antigen concentration process is required, which greatly increases the cost of vaccine preparation. 3. Challenge after immunization can ensure that the immunized individual does not develop the disease or shed the virus. Summary of the Invention

[0006] The purpose of this invention is to provide a highly immunogenic H6 subtype avian influenza virus strain, Alphainfluenzavirus 202601106H6, isolated from clinical samples in Hebei Province. The preservation number of this strain is CCTCC NO: V202657.

[0007] Another objective of this invention is to provide the application of Alphainfluenzavirus 202601106H6 in the preparation of H6 subtype avian influenza vaccines.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] The applicant isolated a highly immunogenic H6 subtype avian influenza virus strain from clinical samples in Hebei Province. Chickens infected with this virus experienced a 30% decrease in egg production and more than 10% mortality. The virus tested positive for both the general influenza virus and the H6 subtype. After homogenization, centrifugation, and filtration of the samples, stable virus was obtained through continuous passage in SPF chicken embryos, exhibiting a high virus titer (EID). 50 ≥ 10 8.0 / 0.1ml, HA potency ≥ 2 9 Analysis of the HA and NA sequences of the virus confirmed that the pathogen is the H6N1 subtype avian influenza virus. This strain has been deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: V202657, classification name: Influenza A virus 202601106H6 Alphaininfluenzavirus, address: Wuhan University, Wuhan, China, deposit date: April 29, 2026.

[0010] The scope of protection of this invention also includes:

[0011] Application of influenza A virus 202601106H6 in the preparation of H6 subtype avian influenza virus vaccine.

[0012] The above-described applications refer to inactivated vaccines.

[0013] Application of influenza A virus 202601106H6 in the preparation of animal models of H6 subtype avian influenza virus infection.

[0014] Application of influenza A virus 202601106H6 in the preparation of drugs for preventing infection with H6 subtype avian influenza virus.

[0015] Application of influenza A virus 202601106H6 in the preparation of drug screening models against H6 subtype avian influenza virus.

[0016] An H6 subtype avian influenza virus vaccine, wherein the vaccine comprises inactivated influenza A virus 202601106H6.

[0017] The vaccines described above also include adjuvants.

[0018] In the vaccine described above, preferably, the adjuvant is Macrol52 white oil.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] Highly representative strain: This strain originated from the front line of the epidemic in Hebei, China, and has excellent matching and representativeness to the H6 subtype virus currently circulating in northern China.

[0021] Excellent immunogenicity: Experimental data show that the inactivated vaccine prepared from this strain can induce high levels of hemagglutination inhibition (HI) antibodies in poultry after vaccination, with uniform antibody levels and long duration of action.

[0022] Outstanding protective efficacy: Challenge protection test shows that the vaccine prepared in this invention can provide 100% immune protection against homologous strains, significantly inhibit clinical symptoms and viral shedding, and has extremely high clinical application value.

[0023] Good production performance: This strain proliferates rapidly and yields high output on chicken embryos, making it suitable for large-scale factory-scale vaccine production and reducing the cost of a single dose of vaccine. Attached Figure Description

[0024] Figure 1 These are the changes in the embryo after the filtrate is inoculated into chicken embryos;

[0025] Among them: left 1 is the control group, left 2 and left 3 are the infection group.

[0026] Figure 2 This is an amplification diagram of nucleic acid extracted from allantoic fluid after reverse mixing. Lane 1 shows the amplification results of the universal HA primer, lane 2 shows the amplification results of the H6 HA specific primer, lane 3 shows the commercially available DL5000 molecular marker, lane 4 shows the amplification results of the N1 NA specific primer, and lane 5 shows the amplification results of the universal influenza M primer.

[0027] Figure 3 These are the necropsy results of chickens that died in the pathogenicity experiment. The leftmost chicken 1 showed a large number of air bubbles and bloody secretions in the trachea and hemorrhage in the tracheal ring. The leftmost chicken 2 showed hemorrhage in the coronary fat of the heart. The leftmost chicken 3 showed necrosis and hemorrhage in the pancreas. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art, and the reagents or materials, unless otherwise specified, are all derived from commercial sources.

[0029] Example 1:

[0030] Isolation, identification and sequence analysis of H6N1 subtype strains

[0031] (1) Source of the pathogen: The pathogen was obtained from clinically dead chickens in a family farm in Hebei Province. The flock consisted of about 20,000 chickens. Before the start of egg production, the chickens had been vaccinated three times with H5 and H7 subtype avian influenza vaccines, and their antibody titers were normal. Around 180 days after the start of egg production, the flock showed abnormalities, including a 30% decrease in egg production and a cumulative mortality rate of more than 10%, with a daily mortality rate exceeding 1%. After dissecting the dead chickens, lungs, livers, and other tissues were collected and frozen for preservation.

[0032] (2) Pathogen isolation and identification: After thawing the frozen tissue, homogenize it three times. Centrifuge the homogenate at 12000 rpm / min at 4℃ for 30 minutes. Filter the supernatant through a 0.22 μm filter membrane and collect the filtrate. Aliquot the filtrate and store at -80℃ for later use. Prepare 9-11 day old SPF chicken embryos. Inoculate 0.2 ml of the prepared filtrate into the allantoic cavity per embryo. Observe continuously for 96 hours after inoculation, discarding any embryos that die within 24 hours. After 96 hours, remove the allantoic fluid and embryo. At this time, the allantoic fluid is clear and transparent, and the embryo shows obvious shrinkage and hemorrhage. The morphology of the embryo is as follows: Figure 1 As shown (left 1 is the control group, left 2 and left 3 are the filtrate receiving groups).

[0033] DNA and RNA were extracted from the harvested allantoic fluid using a commercially available kit. The RNA was then converted to cDNA for identification by fluorescent PCR. The results are shown in Table 1. The harvested allantoic fluid tested positive for H6 avian influenza virus nucleic acid, while other suspected pathogens tested negative. This isolate was named HBHD01.

[0034] Table 1. Nucleic acid detection results of chicken embryo allantoic fluid

[0035] .

[0036] (3) Virus sequencing and typing: Allantoic fluid was reverse-engineered, amplified, and sequenced using universal primers for influenza virus. The amplification results are as follows: Figure 2 As shown, the isolate HBHD01 was positive for the universal HA primer, the H6N1-HA and H6N1-NA specific primers, and the influenza universal primer M, indicating that the isolate HBHD01 is an H6N1 subtype avian influenza virus. The positive fragment was sent to Aoke Biotechnology Co., Ltd. for sequencing and assembly. The sequencing sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. BLAST results showed that the sequence with the closest similarity to the sequence in SEQ ID NO.1 was Influenza A virus (A / chicken / Henan / NL04 / 2025(H6N2)) segment 4 hemagglutininin (HA) gene, Sequence ID: PZ278587.1, with a homology of 99.48%. The sequence with the closest similarity to SEQ ID NO.2 is the Influenza A virus (A / feline / South Korea / SNU1 / 2023(H5N1)) segment 6 neuraminidase (NA) gene, Sequence ID: OR388765.2, with a homology of 97.57%. This result further confirms that HBHD01 is an H6N1 subtype avian influenza virus.

[0037] This strain has been deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: V202657, classification name: Influenza A virus 202601106H6 Alphainfluenzavirus, address: Wuhan University, Wuhan, China, deposit date: April 29, 2026.

[0038] In this invention, the influenza A virus (202601106H6) is abbreviated as HBHD01.

[0039] Example 2:

[0040] Pathogenicity test of H6N1 subtype strain HBHD01

[0041] Ten 35-day-old SPF chickens and ten 68-day-old SPF chickens were randomly selected and injected intravenously with 0.5 ml of HBHD01 allantoic fluid (10 chickens). 8 EID 50 ( / ml), while 5 chickens were additionally selected as blank controls in each group and injected with the corresponding volume of SPF chicken allantoic fluid. The experimental group and the control group were observed continuously in an isolator for 7 days, and the results are shown in Table 2.

[0042] Table 2. Pathogenicity test of H6N1 subtype strain HBHD01

[0043] .

[0044] The mortality rate was 10 / 10 in the young chickens infected, and 2 / 10 in the older chickens infected. Necropsy of the dead chickens revealed numerous air bubbles and bloody secretions in the trachea, and hemorrhage around the tracheal rings. Figure 3 Left 1), coronary fatty hemorrhage of the heart ( Figure 3 Left 2), pancreatic necrosis and hemorrhage ( Figure 3 (Left 3), which is consistent with typical symptoms of influenza infection.

[0045] Therefore, the strain of the present invention can be used to prepare an animal model of infection with the H6N1 subtype strain, and the model can be used to screen drugs against the H6N1 subtype strain.

[0046] Example 3:

[0047] Preparation of H6N1 subtype vaccine:

[0048] HBHD01 was continuously passaged in SPF chicken embryos, and the allantoic fluid of the 5th generation was selected as the seed virus. Sterility, purity, toxicity, and shelf-life tests were performed according to the requirements of the Chinese Veterinary Pharmacopoeia. Samples that passed the tests were aliquoted and cryopreserved, and named HBHD01 P1. The hemagglutination titer of the seed virus was determined to be 2^10, and the seed virus titer was 10. 9 EID 50 / 0.1 ml

[0049] HBHD01 P1 was inoculated into 9-11 day old chicken embryos at a 1:100 dilution with physiological saline and incubated at 37°C for 72 hours. Embryos that died within 24 hours were discarded, and allantoic fluid was harvested from the remaining embryos. The hemagglutination titer was measured to be 2^10, and the titer was 10. 9 EID 50 / 0.1 ml, dilute the antigen titer to 0.25 × 10⁻⁶ with PBS buffer. 9 EID 50 / 0.1 ml, add formaldehyde to a final concentration of 0.3% for inactivation, and the inactivation time is 72 h. Take the inactivated antigen, dilute it with PBS to adjust the antigen content, and use it as the aqueous phase. Take another 96 parts of Macrol52 white oil, heat it to 100℃, then add 4 parts of Span-80, mix thoroughly, and autoclave at 121℃ for later use as the oil phase. Pour 3 parts of the oil phase into an emulsification tank and stir, then slowly add 2 parts of the aqueous phase, stir thoroughly to emulsify, and dispense to obtain the finished vaccine product with a final antigen content of 10. 8 EID 50 / 0.1ml. Perform sterility and inactivation tests on the finished vaccine product.

[0050] Example 4:

[0051] Study on the immunogenicity of H6N1 subtype vaccine

[0052] Ten SPF chickens aged 28-35 days were randomly selected and each was subcutaneously injected with 0.3 ml of the HBHD01 vaccine prepared in Example 3 into the neck and back. They were then housed in isolators in the Keqian Biological Animal House. Twenty-one days after vaccination, blood was collected from five control chickens under identical conditions, and serum was separated. HI antibodies were measured using standard antigens for avian influenza virus H6 and H9 subtypes. The results are shown in Table 3. The results indicate that all HBHD01-immunized chickens were positive for H6 antibodies, and the HI titers were all greater than 1:2. 6 The H9 antibody test was negative.

[0053] Table 3. Detection of antibody levels in chickens after immunization

[0054] .

[0055] Ten SPF chickens aged 28-35 days were randomly selected, and each chicken was subcutaneously injected with 0.3 ml of the HBHD01 vaccine prepared in Example 3 in the back of its neck. They were then housed in isolators in the Keqian Biological Animal House. Twenty-one days after vaccination, along with five control chickens under identical conditions, each chicken was intravenously injected with 0.2 ml of HBHD01 virus solution (containing 2×10⁻⁶ HBHD01 virus cells). 6 EID 50 Low pathogenic avian influenza viruses, represented by H9 and H6 subtypes, typically do not cause acute death or significant lesions in poultry after infection. In this invention, both the experimental and control groups survived after challenge. On the 5th day after challenge, throat and cloacal swabs were collected from each chicken and inoculated into the allantoic cavity of 9-11 day old SPF chicken embryos. The samples were tested according to the efficacy test of the avian influenza immune challenge method given in the Chinese Veterinary Pharmacopoeia. The results showed that 4 / 5 of the control group and 1 / 10 of the immune group were positive for virus isolation.

[0056] This result indicates that the prepared HBHD01 H6N1 subtype vaccine can effectively protect against the attack of H6 subtype avian influenza virus.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An isolated influenza A virus (Alphainfluenzavirus), the preservation number of which is CCTCC NO: V202657.

2. The use of the influenza A virus as described in claim 1 in the preparation of an H6 subtype avian influenza virus vaccine.

3. The application according to claim 2, wherein the vaccine is an inactivated vaccine.

4. The application of the influenza A virus according to claim 1 in the preparation of an animal model of H6 subtype avian influenza virus infection.

5. The use of the influenza A virus according to claim 1 in the preparation of a drug for preventing infection with H6 subtype avian influenza virus.

6. The application of the influenza A virus as described in claim 1 in the preparation of a drug screening model for the H6 subtype avian influenza virus.

7. An H6 subtype avian influenza virus vaccine, said vaccine comprising inactivated influenza A virus as described in claim 1.

8. The vaccine according to claim 7, wherein the vaccine further comprises an adjuvant.

9. The vaccine according to claim 8, wherein the adjuvant is Macrol52 white oil.

Images