Guangxi H9N2 avian influenza virus strain and application thereof

Abstract

The invention discloses a Guangxi H9N2 avian influenza virus strain, the Guangxi H9N2 avian influenza virus strain is named as avian influenza virus A / chick / China / CWM / 2019 (H9N2), and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.45210; the invention also discloses an application of the Guangxi H9N2 avian influenza virus strain in preparation of vaccines. According to the invention, the variation condition of the Guangxi chicken source H9N2 avian influenza virus and other isolated strains is clarified; the protection rate of the CK / GX / CWM / 19 to homologous strains reaches 100%, the protection rate of the CK / GX / CWM / 19 to heterologous strains reaches 80%, and the virus strains can be used as candidate strains of H9 subtype avian influenza vaccines through virus attack immune protection effect analysis.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a Guangxi H9N2 avian influenza virus strain and an application thereof. Background Art

[0002] Avian influenza virus (AIV) is an enveloped single-stranded negative-sense RNA virus with a genome composed of 8 different independent segments. It is genetically unstable and prone to gene recombination and mutation. H9N2 subtype AIV infects chickens and mainly causes respiratory, digestive and reproductive system diseases in chickens, which in turn causes adverse effects such as slow growth and reduced egg production in chickens. It is one of the main subtypes that currently affect the healthy development of my country's poultry industry. H9N2 subtype AIV is the main subtype of AIV currently present in chickens in my country. It often causes respiratory symptoms, reduced egg production in laying hens, and makes chickens susceptible to secondary severe respiratory diseases, affecting poultry production performance; broiler chickens are infected and cause immunosuppression, which is prone to secondary infection and mixed infection, causing huge economic losses to the broiler breeding industry.

[0003] The H9N2 subtype avian influenza virus is widely prevalent in poultry around the world, causing huge economic losses, and cross-species infection of humans, pigs and other animals, which has important public health significance. It is particularly noteworthy that it continues to provide internal gene fragments for newly emerging H5N6, H7N9, H10N8, H3N8 and other subtype avian influenza viruses, posing a huge threat. Although the H9N2 subtype avian influenza inactivated vaccine has played a decisive role in the prevention and control of the disease, the H9N2 subtype avian influenza virus continues to undergo antigenic mutations, which has challenged the prevention and control effect of the vaccine. Vaccination is still one of the most effective means to prevent the occurrence and spread of AI. However, with the continuous mutation and evolution of AIV, the vaccine strain is not highly matched with the current epidemic strain, and the inactivated vaccine cannot provide broad protection. Actively conducting epidemiological surveys, closely tracking the molecular evolution and antigenic variation of AIV, and selecting H9N2 avian influenza virus vaccine strains with good immune protection can ensure the good immune effect of the vaccine. The avian influenza virus mutates very quickly, so timely discovering new strains, studying their characteristics, and ultimately screening out strains that can be used as vaccine strains is one of the most important ways to ensure the effectiveness of the avian influenza vaccine. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problem that the avian influenza virus mutates quickly in the prior art and the existing vaccines cannot keep up with the effective prevention and control measures, and to provide data and lay a foundation for the molecular epidemiological research and vaccine development of H9N2 AIV in Guangxi.

[0005] To achieve the above purpose, the technical solution provided by the present invention is as follows:

[0006] The present invention provides a Guangxi H9N2 avian influenza virus strain, named as avian influenza virus A / chicken / China / CWM / 2019 (H9N2) (abbreviated as CK / GX / CWM / 19), and provides a full genome sequence of the strain; its deposit number is CGMCC No.45210, and it was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on June 20, 2022, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The difference in genetic evolution between the strain HA and the common H9N2 AIV reference strain and the commonly used vaccine strain is clarified through HA gene genetic evolution analysis, and it is found that it is different from the commonly used vaccine strain through antigen difference research, and it is found that it has cross-immune protection effect on different strains in animal immune protection experiments, thereby providing data for the prevention and control research of Guangxi H9N2 AI.

[0007] The use of the Guangxi H9N2 avian influenza virus strain as described above in the preparation of vaccines.

[0008] The use of the Guangxi H9N2 avian influenza virus strain as described above in the preparation of a vaccine for preventing and treating the H9N2 avian influenza virus.

[0009] Preferably, the vaccine is an inactivated vaccine.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The invention clarifies the antigenic variation of Guangxi chicken-derived H9N2 avian influenza virus and other isolated strains; after the strain is prepared into an inactivated vaccine, 4W SPF chickens are immunized, and through the analysis of the immune protection effect of attacking homologous and heterologous H9 influenza wild strains, the protection rate of CK / GX / CWM / 19 against homologous strains reaches 100%, and the protection rate against heterologous strains is 80%, which proves that the virus strain can be used as a candidate strain of H9 subtype avian influenza vaccine.

[0012] Storage Information

[0013] The avian influenza virus A / chicken / China / CWM / 2019 (H9N2) was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on June 20, 2022, with the deposit number CGMCC No.45210. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the genetic evolution tree of HA gene; “■” represents the isolate A / chicken / China / CWM / 2019 (H9N2); “▲” represents the SS strain, a commonly used vaccine strain in China. Figure 2It is the antibody level of the oil-emulsion inactivated vaccine made of avian influenza virus A / chicken / China / CWM / 2019 (H9N2) strain and the commercial vaccine SS strain after immunization of SPF chickens. DETAILED DESCRIPTION

[0015] The following is a detailed description in conjunction with the accompanying drawings and specific embodiments, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. The raw materials and reagents used in the examples are all commercially available unless otherwise specified. A / chicken / Guangxi / CX / 2013 (H9N2) (abbreviated as CK / GX / CX / 13) was isolated and stored in the laboratory of the Guangxi Zhuang Autonomous Region Veterinary Research Institute.

[0016] Example 1

[0017] Virus isolation and purification

[0018] The diseased chicken samples were collected and ground with saline in proportion (sample and saline were added in a mass ratio of 1:3). After repeated freezing and thawing for 3 times, the samples were centrifuged at 8000r / min for 5 minutes, and the supernatant was filtered with a 0.22μm disposable filter. The supernatant was inoculated into 10d SPF chicken embryos by allantoic cavity inoculation, incubated at 37℃ for 96h, and the allantoic fluid of dead and non-dead chicken embryos was collected. The virus proliferation effect was identified by hemagglutination test (HA) and hemagglutination inhibition test (HI). Among them, the samples with positive HI test results were cloned and purified for 3 consecutive rounds by limiting dilution method. The purified samples were cultured and ultracentrifuged and packaged in cryopreservation tubes and stored in a -80℃ refrigerator for later use.

[0019] Genome RT-PCR amplification, sequencing and identification

[0020] The viral genomic RNA in the chicken embryo allantoic fluid stored at -80°C was extracted using an RNA extraction kit. At the same time, amplification primers were synthesized according to the method of Hoffmann et al. (2001) (see Table 1), and the full gene fragment of the viral genome was amplified using the one-step method according to the instructions of the One Step RT-PCR Kit. The obtained PCR product was purified by gel excision, connected with the pMD18-T vector and transformed into DH5α competent cells. After expanded culture, a single colony was picked for PCR identification. The bacterial solution with positive identification results was sent to Shenzhen BGI Genomics Co., Ltd. for sequencing. The full gene sequence is shown in the sequence table from 1 to 8.

[0021] Identification: The sequencing results were searched by BLAST and had the highest homology with the HA and NA genes of the H9N2 subtype avian influenza virus in NCBI. Therefore, according to the standard nomenclature of avian influenza, the strain was named avian influenza virus A / chicken / China / CWM / 2019 (H9N2) strain, abbreviated as CK / GX / CWM / 19.

[0022] Table 1. Primers for amplifying the full-length genome of avian influenza virus

[0023]

[0024]

[0025] Example 2

[0026] The H9 subtype AIV obtained after purification and identification in Example 1 was subjected to the following various operations to determine some of its biological characteristics:

[0027] 1. Genetic evolution analysis

[0028] The HA gene sequence of A / chicken / China / CWM / 2019 (H9N2) is shown in the specification. At the same time, the primers designed by Hoffmann et al. (2001) to amplify the HA gene fragment of AIV were used for primer synthesis (see Table 1), and the H9 subtype AIV HA gene fragments were amplified. The HA gene sequence determination and genetic evolution analysis of the virus showed that its HA gene belongs to the Y-280-like lineage, which is far away from the commonly used vaccine strains and is in a different evolutionary branch. The specific gene evolution tree is shown in Figure 1 As shown (the analysis software used is DNAstar7.0 and MEGA11.0), Figure 1 The “■” A / chicken / Guangxi / CWM / 2019(H9N2) refers to the isolate A / chicken / China / CWM / 2019(H9N2). The name is different due to the preservation process.

[0029] 2. The virus can grow at high titer in chicken embryos, and the hemagglutinin titer is stable at 2 10 about

[0030] EID 50 Determination: A / chicken / China / CWM / 2019(H9N2) virus was diluted 10 times, and 5 SPF chicken embryos were inoculated with each dilution. The eggs were incubated at 35℃ for 48 hours. The hemagglutination activity of the allantoic fluid of the infected chicken embryos was measured to determine whether they were infected. The EID was calculated using the Reed-Muench method. 50The embryonic infectious dose (EID) of A / chicken / China / CWM / 2019(H9N2) 50 ) is 10 -7.8 / ml, the results showed that A / chicken / China / CWM / 2019(H9N2) can easily proliferate in chicken embryos, which is one of the conditions for being a candidate strain for a vaccine.

[0031] 3. The virus can produce higher antibodies in SPF chickens

[0032] After the A / chicken / China / CWM / 2019(H9N2) virus was inactivated with β-propiolactone, it was evenly mixed with white oil (volume ratio 3:7) to make an oil emulsion inactivated vaccine, which was used to immunize 4-week-old SPF chickens (0.3 mL was injected subcutaneously per chicken). After 3 weeks, serum was collected to detect the H9 antibody titer in the serum. The antibody reached a high titer 3 weeks after immunization, that is, the antibody titer of CK / GX / CWM / 19 reached about 7 (lg2) in the 3rd week after immunization. Therefore, the inactivated oil vaccine developed with the CK / GX / CWM / 19 virus strain as the antigen can easily induce an immune response in the chickens after immunizing SPF chickens.

[0033] Example 3

[0034] Virus HA antigenicity analysis

[0035] Experimental methods:

[0036] 1. Preparation of H9 single factor serum

[0037] Oil emulsion inactivated vaccines were prepared from 12 strains of H9N2 subtype AIV isolated from 2013 to 2020 (one of which was the strain CK / GX / CWM / 19 of the present invention) and the commonly used vaccine reference strain SS strain (CK / GD / SS / 94) (each strain is shown in Table 2). 4W-old SPF chickens were immunized with the above-mentioned oil emulsion inactivated vaccine, and single-factor serum was prepared in SPF chicken isolation feeders for cross-HI tests between different strains to detect whether the HA antigen spectrum of CK / GX / CWM / 19 virus is broad-spectrum; two immunizations were performed at an interval of 2W, and the corresponding chicken whole blood was collected 14d after the second immunization to prepare positive serum for cross-HI tests.

[0038] 2. Cross-HI test between different strains

[0039] Using 13 virus strains as antigens, 4 units of each virus were prepared and cross-HI tests were performed with 13 single-factor sera to detect the HA antigen reactivity of the H9N2 virus. The experiment was repeated three times and the average value of the three experiments was taken (Table 2).

[0040] The experimental results in Table 2 above were used for R value analysis to determine the correlation between the above H9N2 virus HA antigens. The criteria for determining antigen correlation are as follows:

[0041] Correlation between antigens

[0042] Antigenic differences among R-2 strains

[0043] r1-HI titer of virus A against serum B / HI titer of virus A against serum A

[0044] r2-HI titer of virus B against serum A / HI titer of virus B against serum B

[0045] If R=1, it indicates that the antigenicity between the two strains is the same; if 0.67≤R≤1.5, it indicates that there is no significant difference in antigenicity between the two strains; if 0.5≤R<0.67, it indicates that there is a small difference in antigenicity between the two strains; if R<0.5, it indicates that there is a significant difference in antigenicity between the two strains; the smaller the R value, the greater the difference in antigenicity. The results are shown in Table 3:

[0046] Table 2 Interactive HI antibody titers of H9 subtype AIV isolates and vaccine strains

[0047]

[0048] Table 3 Antigenic correlation coefficient R values ​​between strains

[0049]

[0050]

[0051] HA antigen correlation analysis

[0052] As can be seen from Table 3, the HA antigen correlation between CK / GX / CWM / 19 virus and the other 12 H9N2 virus strains is greater than 0.53, indicating that the virus has a good correlation with the HA antigen of most strains, but its correlation with the HA antigen of the commercial vaccine SS strain is only 0.49, which is significantly different. This shows that the HA antigen of the hemagglutinin protein of CK / GX / CWM / 19 is relatively broad-spectrum and can be used as a candidate strain for H9N2 avian influenza vaccine.

[0053] Example 4

[0054] Analysis of the immune protection effect of vaccine strains

[0055] 1. Vaccine preparation and cross-immunization

[0056] The isolated strain CK / GX / CWM / 19 was used to prepare an oil emulsion inactivated vaccine, which was used to immunize two groups of 4W SPF chickens, with 10 chickens in each immunization group and 6 chickens in the control group (the control group was not immunized). After 21 days of immunization, the homologous and heterologous isolated strains were used to infect the nasal cavity of 10 SPF chickens in each immunization group and 6 SPF chickens in the blank control group, respectively. The challenge dose was 10 6 EID 50 .

[0057] The results are as follows: Group I was challenged with CK / GX / CWM / 19 at a dose of 100 μL (100 μL containing 10 6 EID 50 ), Group II was challenged with A / chicken / Guangxi / CX / 2013(H9N2)(abbreviated as CK / GX / CX / 13)(isolated and stored in our laboratory) at a dose of 100 μL (nasal drops, containing 10 6 EID 50 ). The throat cotton swab and cloaca cotton swab of each chicken in the experimental group and the control group were collected at 1, 3, 5 and 7 days after the control group was infected, and immediately placed in a -80℃ refrigerator, and the virus was titrated with 9-10d SPF chicken embryos. The mental state and diet of the chickens in the experimental group and the control group were observed every day. Each cotton swab was diluted 10 times in series, and 3 SPF chicken embryos were inoculated at each dilution. The incubation continued at 37℃ for 48h. The HA titer of the infected chicken embryo allantoic fluid was measured to determine whether it was infected, and the virus content was calculated using the Reed-Muench method.

[0058] 2. Antibody test results 3 weeks after immunization

[0059] The CK / GX / CWM / 19 strain was made into an oil emulsion inactivated vaccine and then immunized with 4-week SPF chickens. Serum was collected 21 days after immunization to detect antibodies. The results showed that the H9 antibody titer of the chickens in the experimental group was about 8, as shown in Table 4.

[0060] Table 4 Antibody detection results of serum collected from SPF chickens 21 days after immunization with inactivated vaccine CK / GX / CWM / 19

[0061]

[0062]

[0063] 3. Immune effect of H9N2 avian influenza oil emulsion inactivated vaccine on SPF chickens

[0064] Cotton swabs were collected on days 1, 3, 5 and 7 after challenge and virus titration was performed on 9-day SPF chicken embryos. The specific results are shown in Table 5. Within 7 days after challenge, the throat and cloaca of the 6 chickens in the control group excreted toxins, and the amount of toxins excreted on days 5 and 7 was lower than that on day 3; the protection rate of the immunized group against homologous strains reached 100%, and the protection rate against heterologous strains was 80%.

[0065] 4. The safety effect of H9N2 avian influenza oil emulsion inactivated vaccine on SPF chickens. The virus challenge began 21 days after immunization. After the virus challenge, neither the experimental group nor the control group showed obvious clinical symptoms, which proved that the vaccine is safe for chickens.

[0066] Table 5 Immune protection test of H9 avian influenza oil emulsion inactivated vaccine on SPF chickens

[0067]

[0068] In Table 5, control refers to the blank control group.

[0069] 5. Monitoring of antibody levels after immunization

[0070] The antibody levels of CK / GX / CWM / 19 strain oil emulsion inactivated vaccine and commercial vaccine SS strain after immunization of SPF chickens Figure 2 As shown. After 2 weeks of immunization with the inactivated vaccine made of CK / GX / CWM / 19 strain and the commercial vaccine SS strain, the antibody level of SPF chickens reached above 7log2. After the 4th week of immunization, the antibody HI reached the maximum value. The oil emulsion inactivated vaccine made of CK / GX / CWM / 19 strain can reach a maximum of 10log2, while the maximum of the commercial vaccine of SS strain is 11log2. The antibody level of SPF chickens in the CK / GX / CWM / 19 immunization group began to decline slowly after 10 weeks of immunization. The antibody level of SPF chickens immunized with the CK / GX / CWM / 19 strain inactivated vaccine can still be maintained above 7log2 at 24 weeks. The antibody level of the control group was always below 1log2, and the control was established. It can be seen that the oil emulsion inactivated vaccine made of CK / GX / CWM / 19 strain can maintain a relatively high antibody level in SPF chickens within 24 weeks after immunization. Although the antibody level after immunization with the oil-emulsion inactivated vaccine made from the CK / GX / CWM / 19 strain differs by 1-2log2 from that of the commercial vaccine of the SS strain, it always maintains a high immune level and has good immunogenicity and immune protection effect. It can be used as a vaccine candidate strain.

[0071] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A strain of H9N2 avian influenza virus from Guangxi, Features: The Guangxi H9N2 avian influenza virus strain was named avian influenza virus A / chicken / China / CWM / 2019 (H9N2), and the preservation number is CGMCC No.45210.

2. Use of the Guangxi H9N2 avian influenza virus strain as claimed in claim 1 in preparing a vaccine.

3. Use of the Guangxi H9N2 avian influenza virus strain as claimed in claim 1 in preparing a vaccine for preventing and treating H9N2 avian influenza virus.

4. The use according to claim 2 or 3, Features: The vaccine is an inactivated vaccine.

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