CN113913395B - Artificial recombinant H5N8 influenza virus, and preparation method and application thereof - Google Patents

Abstract

The invention discloses an artificial recombinant H5N8 influenza virus and a preparation method and application thereof. The artificially recombined H5N8 influenza virus HAs high pathogenicity avian influenza virus A/swan/Shanxi/4-1/2020(H5N8) surface antigen Hemagglutinin (HA) and Neuraminidase (NA) genes, but HA cleavage sites present molecular characteristics (-RETR-) of typical low pathogenicity avian influenza virus, HAs 6 internal genes of PB2, PB1, PA, NP, M and NS of influenza virus chick embryo high titer adaptive strain A/PR/8/34(H1N1), HAs strain numbers of H5-Re14, and HAs a CCTCC NO: v202160. The chicken embryo adaptability is good, and the virus growth titer can be improved by more than 9 times compared with that of wild virus; the vaccine strain is used for producing the vaccine, so that the production cost of the vaccine can be greatly reduced, and the quality of the vaccine is improved.

Description

Artificial recombinant H5N8 influenza virus and its preparation method and application

technical field

The invention relates to the technical field of reverse genetics technology and animal infectious diseases, in particular to an artificially recombined and weakened H5N8 subtype recombinant influenza virus and a preparation method and application thereof.

Background technique

Avian influenza virus (AIV) is a segmented negative-strand RNA virus that can broadly infect birds and mammals. AIV is classified into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes based on the surface proteins of the virus. Among them, H5 and H7 subtypes of AIV can cause outbreaks in poultry or wild birds, resulting in significant economic losses, and can also infect humans and cause public health hazards. China has developed a series of whole virus inactivated vaccines, which have successfully controlled H5N1 since 2004 and H7N9 subtype avian influenza since 2017.

In order to deal with the possible occurrence of poultry H5N8 epidemic situation and public health hazards in my country, the present invention conducts the development of artificially recombined H5N8 avian influenza vaccine strains.

The ideal influenza virus vaccine strain should have good antigenic matching with the circulating strain, no pathogenicity to chicken embryos and high titer growth of chicken embryos. However, it is difficult to isolate influenza strains having the above conditions as vaccine strains from nature.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide an artificially reconstituted H5N8 influenza virus and a preparation method and application thereof.

The technical scheme of the present invention is as follows: the recombinant H5N8 avian influenza virus uses the highly pathogenic H5N8 subtype avian influenza virus A/swan/Shanxi/4-1/2020 (H5N8) as the surface antigen hemagglutinin (HA) and neural Aidase (NA) gene donor, but the HA cleavage site has the typical molecular characteristics of low pathogenic avian influenza virus (-RETR-); adapted to the high titer of influenza virus chicken embryo strain A/PR/8/34 ( The PB2, PB1, PA, NP, M and NS genes of H1N1) are the donors of 6 internal genes. Through reverse genetic manipulation, a recombinant H5N8 avian influenza virus was obtained by artificial recombination, which was named as influenza A virus A/Harbin/H5-Re14/2021 (H5N8) (referred to as H5-Re14 strain), deposited in the China Center for Type Culture Collection, located in Wuhan University, Wuhan, China, its deposit number is CCTCC NO: V202160, and the preservation time is August 3, 2021.

The present invention selects 6 internal genes of the laboratory chicken embryo-adapted strain A/PR/8/34 (H1N1) of influenza virus that can grow to a very high titer in chicken embryos as backbone genes, and selects the 6 internal genes isolated in 2020. The HA and NA genes of the highly pathogenic avian influenza virus strain A/swan/Shanxi/4-1/2020 (H5N8) were used as surface genes, and the attenuated recombinant virus H5-Re14 was prepared by artificial construction method using reverse genetics The recombinant strain will be used as a vaccine strain for H5N8 influenza.

Experiments have confirmed that the H5-Re14 strain virus has the same antigenicity as the H5N8 avian influenza virus strain A/swan/Shanxi/4-1/2020 (H5N8) in China. The attack of Shanxi/4-1/2020 (H5N8) produces a good specific protection effect. The virus contains 6 internal genes of the high-titer influenza virus-adapted strain A/PR/8/34 (H1N1), so it has good chicken embryo adaptability, and the virus growth titer can be increased by 9 times compared with the wild virus The above; using this as a vaccine strain to produce a vaccine can greatly reduce the cost of vaccine production and improve the quality of the vaccine.

Further, the present invention also proposes the application of the recombinant H5N8 avian influenza virus (H5-Re14 strain) in preparing a medicine for preventing diseases caused by the H5N8 influenza virus.

Further, the H5N8 influenza virus is the A/swan/Shanxi/4-1/2020 (H5N8) strain.

Further, the present invention also proposes a method for constructing the described recombinant H5N8 avian influenza virus, the method comprising:

(1) Using HA gene-specific amplification and mutation primers, RT-PCR method was used to amplify the HA1 and HA2 gene fragment, while amplifying the HA1 fragment, mutates the amino acid at the cleavage site of the HA gene from -REKRRKR- to -RETR-, making it mutated from the molecular characteristics of highly pathogenic avian influenza virus to a typical low pathogenic avian influenza virus. Molecular characteristics; then use the overlap extension reaction to amplify the HA fragment whose cleavage site is the molecular characteristics of typical low pathogenic influenza virus, insert it into the pBD plasmid, and construct the bidirectional transcription vector of the recombinant HA gene;

(2) Using RT-PCR method, amplify the NA gene whole gene fragment of H5N8 subtype avian influenza virus A/swan/Shanxi/4-1/2020 (H5N8), insert into pBD plasmid, construct recombinant NA gene bidirectional transcription vector ;

(3) Two recombinant pBD bidirectional transcription vector plasmids of HA and NA genes constructed as described in (1) and (2) and PB2 containing influenza virus chicken embryo high titer adaptation strain A/PR/8/34 (H1N1) , 6 pBD bidirectional transcription vector plasmids of PB1, PA, NP, M, NS genes are mixed, and inoculated with 293T cells together with transfection reagent, and the cells and supernatant harvested after culture are inoculated into chicken embryos to obtain H5N8 with HA activity Avian influenza rH5N8-2/6 recombinant virus strain. The obtained recombinant virus HA and NA are from the highly pathogenic H5N8 influenza virus domestic isolate A/swan/Shanxi/4-1/2020 (H5N8), but the HA cleavage site is typical of low pathogenic avian influenza virus molecular characteristics (-RETR-), 6 internal genes PB2, PB1, PA, NP, M and NS are derived from the high titer-adapted strain A/PR/8/34 (H1N1) of influenza virus in chicken embryos.

Further, the primer pair for amplifying the HA1 gene is shown in SEQ ID No. 9 and SEQ ID No. 10, wherein SEQ ID No. 10 is the mutation primer of the HA cleavage site; the primer pair for amplifying the HA2 gene is as shown in SEQ ID No. 11 and SEQ ID No. 12.

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

1. H5-Re14 has the same antigenicity as my country's H5N8 avian influenza virus strain A/swan/Shanxi/4-1/2020 (H5N8). -1/2020 (H5N8) attack produces good specific protection. The virus contains 6 internal genes of the high-titer influenza virus-adapted strain A/PR/8/34 (H1N1), so it has good chicken embryo adaptability, and the virus growth titer can be increased by 9 times compared with the wild virus The above; using this as a vaccine strain to produce a vaccine can greatly reduce the cost of vaccine production and improve the quality of the vaccine.

2. By artificially modifying the cleavage site of the HA gene, the H5-Re14 strain virus has the molecular characteristics of a typical low pathogenic influenza virus. The H5-Re14 strain recombinant virus has no pathogenicity to chicken embryos and chickens, and has high biological safety. .

Description of drawings

Fig. 1 is the gel electrophoresis image of the gene amplified by RT-PCR. In the figure, A is the cDNA of A/swan/Shanxi/4-1/2020 (H5N8) strain as the template. HA and NA gene fragments, lanes 1 to 4, respectively, M is DNAMarker 2000; B is the 8 gene fragments of recombinant virus amplified by RT-PCR using the cDNA of recombinant H5N8 virus as a template, lanes 1 to 8 are respectively For HA, NA, PB2, PB1, PA, NP, M, NS gene fragments, M is DNA Marker 2000.

Figure 2 shows the sequence before and after mutation of the HA gene cleavage site region of the H5-Re14 virus.

Figure 3 is a growth curve of recombinant H5-Re14 virus and wild-type H5N8 influenza virus A/swan/Shanxi/4-1/2020 (H5N8).

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from commercial channels unless otherwise specified.

Experimental Materials

1. Virus strain

A/swan/Shanxi/4-1/2020 (H5N8) (SW/SX/4-1/20 for short) was isolated from sick and dead swans in Shanxi, my country in 2020. Laboratory isolation, identification and preservation, the strain is recorded in the document: "Research on the immune efficacy of the current recombinant avian influenza virus (H5+H7) trivalent inactivated vaccine on recent H5 and H7 virus antigenic variants", Liu Yanjing et al., " Chinese Journal of Preventive Veterinary Medicine, published online by CNKI in September 2021. The applicant guarantees that the biological material will be available within 20 years from the filing date.

2. SPF chicken embryos and cells

SPF chicken embryos and SPF chickens were purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/National Avian Laboratory Animal Resource Bank, and 293T cells (human embryonic kidney cells, SCSP-502) were purchased from the Cell Bank of the Type Culture Collection, Chinese Academy of Sciences.

3. Plasmid vectors

pBD vectors (Zejun Li, Hualan Chen, Peirong Jiao, Guohua Deng, Guobin Tian, Yanbing Li, Erich Hoffmann, Robert G. Webster, Yumiko Matsuoka, and Kangzhen Yu. Molecular Basis of Replication of Duck H5N1 Influenza Viruses in aMammalian Mouse Model.J .Virol.2005, 79:12058-12064) was constructed by researcher Chen Hualan, and the unidirectional transcription plasmid vector pPolIsapIRib contains the polymerase I promoter-SapI cloning site-The XbaI restriction fragment of the mouse ribozyme sequence was inserted in reverse. The XbaI site of pCI (Promega) plasmid forms RNA polymerase II promoter (CMV) → viral RNA transcription termination signal sequence Rib → influenza virus genome cDNA (5'→3') → human RNA polymerase I promoter → mRNA The bidirectional transcription expression plasmid vector composed of the transcription termination PolyA signal sequence (SV40) can be used to simultaneously transcribe the negative strand vRNA and the positive strand mRNA of influenza virus. pBD-PB2, pBD-PB1, pBD-PA, pBD-NP, pBD-M and pBD-PB2, pBD-PB1, pBD-PA, pBD-NP, pBD-M and The pBD-NS vector was identified and preserved by the National Avian Influenza Reference Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Guobin Tian, Suhua Zhang, Yanbing Li, ZhigaoBu, Peihong Liu, Jiyong Zhou, Chengjun Li, Jianzhong Shi, Kangzhen Yu, Hualan Chen. Protective efficacy in chickens, geese and ducks of an H5N1 inactivatedvaccine developed by reverse genetics. Virology, 2005, 341:153-162).

4. Main reagents

Viral RNA extraction kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; Reverse Transcription Kit (ReverTraAce RT Kit) was purchased from Japan Toyobo Company; Ex-taq enzyme was purchased from Takara Company; PCR product purification kit (CYCLE-PURE- KIT) was purchased from Omega Company; Gel Recovery Kit (Wizard SV Gel and PCR Clean-Up System) was purchased from Promega Company; Sequencing Kit (ABI BigDye Terminator v3.1) was purchased from American Thermo Fisher Company; Plasmid Extraction Kit ( QIAGEN Plasmid Midi Kit) was purchased from QIAGEN Company.

5. Primers

For analysis and comparison, specific primers for the HA1, HA2 and NA genes of the H5N8 influenza isolate SW/SX/4-1/20 (H5N8) were designed and synthesized. The sequences of the synthetic primers are shown in Table 1.

Table 1 HA and NA gene-specific primer sequences of H5N8 avian influenza virus SW/SX/4-1/20 (H5N8)

For analysis and comparison, specific primers for the PB2, PB1, PA, NP, M, and NS genes of A/PR/8/34 (H1N1) were designed and synthesized. The sequences of the synthetic primers are shown in Table 2.

Table 2 Specific primer sequences of 6 internal gene fragments of influenza virus A/PR/8/34 (H1N1)

Example 1: Preparation and identification of recombinant H5N8 influenza virus H5-Re14

1. Construction and identification of two surface genes (HA and NA) of recombinant virus:

The RNA of the H5N8 subtype highly pathogenic avian influenza isolate SW/SX/4-1/20 (H5N8) was extracted and reverse transcribed into cDNA using HA1 fragment-specific primers for the HA gene (HA1-U: atg gag aac ata gta ctt ctt ctt g; HA1-L-: gcc ccg aac agg cct cta gtt tct ctt aga gga cta ttt ctg agc, where HA1-L is a mutant HA cleavage site that is characteristic of low pathogenic virus molecules primers) and HA2 fragment-specific primers (HA2-U: actaga ggc ctg ttc ggg gcg ata gca ggg t; HA2-L: tta aat gca aat tct gca ctg taac), using RT-PCR method to amplify the HA gene respectively HA1 and HA2 fragments (A in Figure 1), and at the same time amplify the HA1 gene fragment, the HA cleavage site is modified by weakening mutation; then HA1 and HA2 are used as templates, HA1-U/HA2-L Overlap extension reaction (SOE-PCR) was performed with phusion high-fidelity polymerase to amplify the HA fragment with the cleavage site typical of low pathogenic avian influenza virus (-RETR-) (Fig. 1, A). It was cloned into pBD vector and constructed into pBD-HA recombinant plasmid.

The mutation primer (HA1-L) of HA gene cleavage site is designed according to the specific sequence of multiple consecutive basic amino acid sites of HA gene, and the amplified HA gene cleavage site is mutated from -REKRRKR- to -RETR-, so that It was mutated from the molecular features of highly pathogenic avian influenza viruses to the typical molecular features of low pathogenic avian influenza viruses (Figure 2).

Then, RT-PCR method was used to amplify the full length of the NA gene of the above-mentioned highly pathogenic H5N8 strain (A in Figure 1), and insert it into the pBD vector according to the above method to construct a pBD-NA recombinant plasmid.

2. Rescue of recombinant virus:

Using the lipofection method, the above two recombinant plasmids of HA and NA genes and pBD-PB2, pBD-PB1, pBD-PA, pBD-NP, pBD-PB2, pBD-PB1, pBD-PA, pBD-NP, Six recombinant plasmids including pBD-M and pBD-NS were simultaneously introduced into monolayer 293T cells. After 48 hours, the cells and supernatant were harvested and inoculated into the allantoic cavity of 9-11-day-old chicken embryos. After 48 hours, the allantoic fluid was harvested and detected. Hemagglutination (HA) activity. The HA positive sample is the rescued H5N8 subtype low pathogenic recombinant virus, and the obtained recombinant virus is named A/Harbin/H5-Re14/2021 (H5N8), the strain number is H5-Re14, and is preserved in Wuhan, China The Chinese Type Culture Collection of Wuhan University, whose deposit number is CCTCC NO: V202160.

3. Sequence Identification of Recombinant Viruses

The RNA of the recombinant virus H5-Re14 was extracted, and 8 fragments of the whole genome of the recombinant virus H5-Re14 were amplified by RT-PCR method. The PCR products were subjected to nucleic acid gel electrophoresis (B in Figure 1), and the gel was recovered and determined The specific sequence of each fragment.

The HA gene sequence of the H5-Re14 strain is shown in SEQ ID No.1, the NA gene sequence of the H5-Re14 strain is shown in SEQ ID No.2, the PB2 gene sequence of the H5-Re14 strain is shown in SEQ ID No.3, and the H5-Re14 strain PB2 gene sequence is shown in SEQ ID No.3. The PB1 gene sequence of the Re14 strain is shown in SEQ ID No.4, the PA gene sequence of the H5-Re14 strain is shown in SEQ ID No.5, the NP gene sequence of the H5-Re14 strain is shown in SEQ ID No.6, and the H5-Re14 strain is shown in SEQ ID No.6. The M gene sequence is shown in SEQ ID No. 7, and the NS gene sequence of the H5-Re14 strain is shown in SEQ ID No. 8.

Using DNAStar software (DNAStar Lasergene V7.1), the determined sequences were compared with the domestic isolates of wild strain H5N8 influenza virus SW/SX/4-1/20 (H5N8) and influenza virus A/PR/8/34 (H1N1) The sequence of the recombinant virus H5-Re14 was compared, and it was found that the HA and NA of the recombinant virus H5-Re14 were derived from the highly pathogenic H5N8 influenza virus domestic isolate SW/SX/4-1/20 (H5N8), but the HA cleavage site showed a typical low pathogenicity. Molecular characterization of pathogenic influenza virus (-RETR-); 6 internal genes, PB2, PB1, PA, NP, M and NS, are derived from chicken embryo highly adaptive influenza virus A/PR/8/34 (H1N1).

4. Determination of the growth curve of recombinant virus H5-Re14

Avian influenza vaccines are usually produced from chicken embryos, and the vaccine strains are required to have good growth characteristics in the inoculated chicken embryos. In this study, the growth characteristics of H5-Re14 strain virus in chicken embryos were studied. The parental virus H5N8 influenza virus strain SW/SX/4-1/20 (H5N8) and the recombinant virus H5-Re14 were inoculated with 20 SPF chicken embryos in each group, and were cultured at the optimum 36°C for 24, 48, and 72 and 96 hours, each 5 chicken embryos cultured at different times were taken for hemagglutination assay, and the growth curves of parental strain SW/SX/4-1/20 (H5N8) and recombinant virus H5-Re14 were detected.

It can be seen from the test results that the growth titer of the recombinant virus (the average HA titer is up to 8.6log2, i.e. 1:388) is higher than that of the parental H5N8 influenza virus domestic isolate SW/SX/4-1/ 20(H5N8) (the highest average HA titer was 5.4log2, i.e. 1:42.2) had a significant increase, and the hemagglutination titer was 9.19 times higher (Fig. 3). This shows that the present invention has prepared a recombinant virus with high growth titer, which will bring huge economic benefits to the subsequent batch production of vaccines.

5. Antigenicity analysis of recombinant virus H5-Re14

Antigenicity is a key indicator of whether a virus can be used as a vaccine strain. In this study, the parental H5N8 influenza virus domestic isolate SW/SX/4-1/20 (H5N8) and the artificial recombinant H5N8 influenza virus were inactivated to prepare oil emulsion inactivated vaccine, and SPF chickens were immunized to prepare antiserum, which was tested by HI test. For antigenicity analysis, HI test method refers to the National Standard for Diagnostic Technology of Highly Pathogenic Avian Influenza (GB) (GB/T118936-2020)). The results of the HI test showed that there was no difference in the cross-HI titer between the parental H5N8 influenza virus domestic isolate SW/SX/4-1/20 (H5N8) and the artificially reconstituted H5N8 influenza virus and the corresponding serum, indicating that the H5N8 virus was recombined into chicken After the embryo-adapted H5-Re14 strain, the virus retained the antigenicity of the parental strain (see Table 3).

Table 3: Antigenicity analysis of parental strain SW/SX/4-1/20 (H5N8) and recombinant virus H5-Re14 strain

6. The virulence test of recombinant virus H5-Re14 strain on chicken embryos and chickens

Low pathogenicity of vaccine strains is a prerequisite for biosafety. 9-11-day-old SPF chicken embryos and 4-8 week-old SFP chickens are often used as models for the pathogenicity assessment of avian influenza viruses.

In order to understand the pathogenicity of the recombinant strain, the present invention firstly uses SPF chicken embryos to evaluate the pathogenicity of the virus. 9-day-old SPF chicken embryos (purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/National Avian Laboratory Animal Resource Bank) were randomly divided into 2 groups of 10 embryos. The first group was inoculated with ^10-4 The virus fluid of recombinant H5-Re14 strain was diluted 10 times, and the second group was inoculated with ^10-4 times diluted virus fluid of parental strain SW/SX/4-1/20 (H5N8) through the allantoic cavity. The death of chicken embryos was observed and recorded within 72 hours after inoculation.

The present invention simultaneously uses SPF chicken to evaluate the pathogenicity and infectivity of the virus. Twenty 6-week-old SPF chickens were randomly divided into 2 groups with 10 chickens in each group. Allantoic fluid of recombinant strain H5-Re14 and parental strain SW/SX/4-1/20(H5N8) were diluted 10 times respectively. After inoculation, 6-week-old SPF chickens were inoculated intravenously. Morbidity and mortality were observed every day after inoculation for 10 consecutive days, and the intravenous inoculation pathogenic index (IVPI) of chickens was calculated. If the IVPI is greater than 1.2, it indicates that the strain is a highly pathogenic strain; if the IVPI is less than 1.2, and the HA cleavage site is a molecular feature of a highly pathogenic avian influenza virus, it is a highly pathogenic avian influenza strain; if If the IVPI is less than 1.2, and the HA cleavage site is a molecular feature of low pathogenic influenza virus, it is a low pathogenic avian influenza strain.

Twenty 6-week-old SPF chickens were randomly divided into 2 groups, 10 in each group. The recombinant strain H5-Re14 and parental strain SW/SX/4-1/20(H5N8) allantoic fluid were diluted to 10 After ⁶ EID ₅₀ /ml, 10 6-week-old SPF chickens were inoculated by nasal route, 0.1ml/chicken. After inoculation, the morbidity and mortality were observed daily for 14 consecutive days. At the same time, throat and cloacal swabs were collected for virus titration to evaluate the ability of the virus to infect chickens.

All test chickens were raised in negative pressure isolators with independent ventilation systems, and all experiments involving H5N8 highly pathogenic virus were carried out in a biosafety level 3 laboratory.

The results of the SPF chicken embryo pathogenic test showed that the chicken embryos inoculated with the parental virus strain SW/SX/4-1/20 (H5N8) virus liquid all died within 26 to 30 hours after inoculation, and the recombinant virus strain H5-Re14 strain was inoculated. All chicken embryos in the virus solution survived within 72 hours after inoculation, indicating that the recombinant virus of H5-Re14 strain is not pathogenic to chicken embryos.

The test results of intravenous inoculation pathogenic index (IVPI) of SPF chickens showed that all chickens inoculated with the parental strain SW/SX/4-1/20 (H5N8) virus liquid became morbid and died within 1 day after inoculation, and their IVPI was 3.0; All chickens inoculated with the recombinant virus strain H5-Re14 strain did not have any adverse reactions within 10 days, and the constructed H5-Re14 strain recombinant virus had an IVPI of 0 (see Table 4). It shows that the recombinant virus of H5-Re14 strain is a low pathogenic strain and has no pathogenicity to chickens.

The results of the nasal infection test of SPF chickens showed that all chickens inoculated with the parental strain SW/SX/4-1/20 (H5N8) virus liquid in the nasal cavity died within 2 to 3 days after inoculation; All chickens with the virus strain had no adverse reactions during the 14-day observation period; all chickens infected with SW/SX/4-1/20 (H5N8) virus died of virus detection in throat and cloacal swabs were positive, while infected The swab samples of chickens with H5-Re14 strain were negative for virus detection on the 3rd and 5th day, indicating that the H5-Re14 strain recombinant virus had no ability to infect and cause chickens (see Table 4).

The above results show that the recombinant virus of H5-Re14 strain has no pathogenicity to chicken embryos and chickens, and has high biological safety.

Table 4 Determination of pathogenicity and infectivity of H5-Re14 strain recombinant virus

*All chickens in this group became ill and died within 2-3 days after challenge, and all dead chickens were detoxified and counted in the results of detoxification on 3 days after challenge; / There was no survival of chickens in this group on 5 days after challenge.

7. Immunogenicity assessment of recombinant virus H5-Re14 strain

The recombinant virus H5-Re14 strain with an HA value of 8log2 was inactivated with 0.2% formaldehyde, and an oil-emulsion inactivated vaccine was prepared with American sornneborn brand-40 (Lytol) white oil as an adjuvant. Ten 3-week-old SPF chickens were used. For model animals, 0.3ml/injection was used for immunization, and 10 non-immunized control chickens were set at the same time. The HI antibody titers in the serum of all chickens were detected on the 21st day after immunization, and SW/SX/4-1/20 (H5N8) was challenged with a dose of 100CLD ₅₀ (CLD50 is the median lethal dose of chickens), and observed for 14 days after the challenge , observe the survival situation, and collect throat and cloaca swabs on the 3rd and 5th days after the challenge, and inoculate chicken embryos to detect the detoxification situation. Swab samples of dead chickens were collected at any time.

The results showed that on the 21st day of immunization, the HI antibody titer of the immunized chicken serum against H5N8 virus was between 7.0log2 and 9.0log2, and the average titer was 8.0log2. The HI antibody titer of the control chicken serum against H5N8 virus was less than 2.0. log2 (HI<2log2 is judged to be negative for HI antibody); 21 days after immunization, the virulent H5N8 subtype avian influenza was challenged with a dose of 100LD ₅₀ via the nasal infection route. The virus detection of the samples were all negative; while the control chickens all died within 5 days after the challenge, and the virus isolation results of the throat and cloacal swab samples of the dead chickens were all positive (see Table 5).

Table 5 H5-Re14 strain recombinant virus preparation vaccine HI antibody after immunization of chickens and survival and detoxification after challenge

*All chickens in this group died of disease within 2 to 4 days after challenge, all dead chickens were tested for detoxification, and the results of detoxification were included in the results of detoxification on the 3rd day after challenge; / The chickens in this group did not survive 5 days after challenge.

The above results show that the artificial recombinant H5N8 influenza virus H5-Re14 has good immunogenicity and can prevent H5N8 subtype avian influenza.

sequence listing

<110> Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin Branch of China Center for Animal Health and Epidemiology)

<120> Artificial recombinant H5N8 influenza virus and its preparation method and application

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1695

<212> DNA

<213> Artificial Sequence

<400> 1

atggagaaca tagtacttct tcttgcaata gttagccttg ttaaaagtga tcagatttgc 60

attggttacc atgcaaacaa ttcgacagag caagttgaca cgataatgga aaagaacgtc 120

actgttacac atgcccaaga catactggaa aaaacacaca acgggaagct ctgtgatcta 180

aatggggtga agcctctgat tttaaaggat tgtagtgtag ctggatggct cctcggaaac 240

ccaatgtgcg acgaattcat cagagtgccg gaatggtcct acatagtgga gagggctaat 300

ccagctaatg acctctgtta cccaggggagc ctcaatgact atgaagaact gaaacacctg 360

ttgagcagaa taaatcattt tgagaagatt ctgatcatcc ccaagagttc atggccaaac 420

catgaaacat cactaggggt gagcgcagct tgtccatacc agggagcgcc ctcctttttc 480

agaaatgtgg tgtggcttat caaaaagaac gatgcatacc caacaataaa gataagctac 540

aataatacca atcgggaaga tctcttgata ctgtggggga ttcatcattc caacaatgca 600

gaagagcaga taaatctcta taaaaaccca accacctaca tttcagttgg aacatcaact 660

ttaaaccaga ggttggtacc aaaaatagct actagatccc aagtaaacgg gcaacgtgga 720

agaatggact tcttctggac aattttaaaa ccggatgatg caatccattt cgagagtaat 780

ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaagggga ctcaacaatt 840

atgaaaagtg gagtggaata tggccactgc aacaccaaat gtcaaacccc agtaggagcg 900

ataaattcta gtatgccctt ccacaacata catcctctca ccattgggga atgccccaaa 960

tacgtgaaat caaacaagtt ggtccttgcg actgggctca gaaatagtcc tctaagagaa 1020

actagaggcc tgttcggggc gatagcaggg tttatagagg gaggatggca gggaatggtt 1080

gatggttggt atgggtacca ccatagcaat gagcagggga gtgggtacgc tgcagacaaa 1140

gaatccaccc aaaaggcaat agatggagtt accaataagg tcaactcaat cattgacaaa 1200

atgaacactc aatttgaggc agttggaagg gagtttaata acttagaaag gaggatagag 1260

aatttgaaca agaaaatgga agacggattc ctagatgtct ggacctataa tgctgaactt 1320

ctagttctca tggaaaacga gaggactcta gatttccatg attcaaatgt caagaacctt 1380

tacgacaaag tcagactaca gcttagggat aatgcaaagg agctgggtaa cggctgtttc 1440

gaattctatc acaaatgcga taatgaatgt atggaaagtg tgagaaatgg gacgtatgac 1500

taccctcagt attcagaaga agcaagatta aaaagagaag aaataagcgg agtgaaatta 1560

gaatcaatag gaacttacca gatactgtca atttattcaa cagcggcgag ttccctagca 1620

ctggcaatca tgatggctgg tctatcttta tggatgtgct ccaatgggtc gttacagtgc 1680

agaatttgca tttaa 1695

<210> 2

<211> 1413

<212> DNA

<213> Artificial Sequence

<400> 2

atgaatccaa atcagaaaat agcgaccatt ggctccatct cattgggact agttgtattc 60

aatgttctac tgcatgcctt gagcatcata ttaatggtgt tagccctggg gaaaagtgaa 120

aacaatggaa tctgcaaggg aactatagta agggaatata atgaaacagt taggatagag 180

aaagtgaccc agtggtacaa cactagtgta gtcgaacatg taccgcattg gaacgagggc 240

gcttatataa acaacaccga accaatatgt gatgtcaagg gctttgcacc tttttccaag 300

gacaacggaa taagaattgg ctccagagga catatttttg tcataaggga gcctttcgtc 360

tcttgttcac ctgtagagtg cagaactttc ttcctcactc agggagctct actcaatgac 420

aaacactcaa atggaacagt gaaggatagg agcccattca gaactctcat gagtgtcgaa 480

gtgggtcaat cacccaatgt gtatcaagca aggtttgaag ctgtagcatg gtcagcaaca 540

gcctgtcatg atggtaagaa atggatgacg attggtgtga cagggccaga ttcgaaagca 600

atagcagtag tccattacgg aggagtgccc actgatattg ttaactcctg ggcaggagac 660

atattacgga ctcaggagtc atcttgtact tgcattcaag gtaattgtta ttgggtaatg 720

actgacggtc catccaatag acaggcgcag tatagaatat acaaagcaaa tcaaggcaaa 780

ataattgacc aagcagatgt cagctttagt ggagggcata ttgaggaatg ctcttgttat 840

ccaaatgatg gtaaagtgga atgcgtatgt agagacaact ggatgggaac taacaggcct 900

gtgctagtta tctcgcctga cctctcttac agggttgggt atttatgtgc gggattgccc 960

agtgacactc caagagggga agatgcccaa tttgtcggtt cgtgcactag tcccatggga 1020

aatcaggggt atggcgtaaa aggtttcggg tttcgacagg gaactgatgt gtggatgggg 1080

cggacaatta gtcgaacctc caggtcaggg tttgaaataa taaggataaa gaatggttgg 1140

acgcagacaa gcaaagaaca gattagaagg caggtggttg ttgataattt gaattggtcg 1200

ggatacagtg ggtctttcac ttaccagta gaattgtctg ggagggaatg tttagtcccc 1260

tgtttttggg tcgaaatgat cagaggcagg ccagaagaaa gaacaatctg gacctctagt 1320

agctccattg taatgtgtgg agttgatcat gaaattgccg attggtcatg gcacgatgga 1380

gctattcttc cctttgacat cgatgggatg taa 1413

<210> 3

<211> 2280

<212> DNA

<213> Artificial Sequence

<400> 3

atggaaagaa taaaagaact aagaaatcta atgtcgcagt ctcgcacccg cgagatactc 60

acaaaaacca ccgtggacca tatggccata atcaagaagt acacatcagg aagacaggag 120

aagaacccag cacttaggat gaaatggatg atggcaatga aatatccaat tacagcagac 180

aagaggataa cggaaatgat tcctgagaga aatgagcaag gacaaacttt atggagtaaa 240

atgaatgatg ccggatcaga ccgagtgatg gtatcacctc tggctgtgac atggtggaat 300

aggaatggac caatgacaaa tacagttcat tatccaaaaa tctacaaaac ttattttgaa 360

agagtcgaaa ggctaaagca tggaaccttt ggccctgtcc attttagaaa ccaagtcaaa 420

atacgtcgga gagttgacat aaatcctggt catgcagatc tcagtgccaa ggaggcacag 480

gatgtaatca tggaagttgt tttccctaac gaagtgggag ccaggatact aacatcggaa 540

tcgcaactaa cgataaccaa agagaagaaa gaagaactcc aggattgcaa aatttctcct 600

ttgatggttg catacatgtt ggagagagaa ctggtccgca aaacgagatt cctcccagtg 660

gctggtggaa caagcagtgt gtacattgaa gtgttgcatt tgactcaagg aacatgctgg 720

gaacagatgt atactccagg aggggaagtg aagaatgatg atgttgatca aagcttgatt 780

attgctgcta ggaacatagt gagaagagct gcagtatcag cagacccact agcatcttta 840

ttggagatgt gccacagcac acagattggt ggaattagga tggtagacat ccttaagcag 900

aacccaacag aagagcaagc cgtggatata tgcaaggctg caatgggact gagaattagc 960

tcatccttca gttttggtgg attcacattt aagagaacaa gcggatcatc agtcaagaga 1020

gaggaagagg tgcttacggg caatcttcaa acattgaaga taagagtgca tgagggatct 1080

gaagagttca caatggttgg gagaagagca acagccatac tcagaaaagc aaccaggaga 1140

ttgattcagc tgatagtgag tgggagagac gaacagtcga ttgccgaagc aataattgtg 1200

gccatggtat tttcacaaga ggattgtatg ataaaagcag ttagaggtga tctgaatttc 1260

gtcaataggg cgaatcagcg actgaatcct atgcatcaac ttttaagaca ttttcagaag 1320

gatgcgaaag tgctttttca aaattgggga gttgaaccta tcgacaatgt gatgggaatg 1380

attgggat tgcccgacat gactccaagc atcgagatgt caatgagagg agtgagaatc 1440

agcaaaatgg gtgtagatga gtactccagc acggagaggg tagtggtgag cattgaccgg 1500

ttcttgagag tcagggacca acgaggaaat gtactactgt ctcccgagga ggtcagtgaa 1560

acacagggaa cagagaaact gacaataact tactcatcgt caatgatgtg ggagattaat 1620

ggtcctgaat cagtgttggt caatacctat caatggatca tcagaaactg ggaaactgtt 1680

aaaattcagt ggtcccagaa ccctacaatg ctatacaata aaatggaatt tgaaccattt 1740

cagtctttag tacctaaggc cattagaggc caatacagtg ggtttgtaag aactctgttc 1800

caacaaatga gggatgtgct tgggacattt gataccgcac agataataaa acttcttccc 1860

ttcgcagccg ctccaccaga gcaaagtaga atgcagttct cctcatttac tgtgaatgtg 1920

aggggatcag gaatgagaat acttgtaagg ggcaattctc ctgtattcaa ctacaacaag 1980

gccacgaaga gactcacagt tctcggaaag gatgctggca ctttaaccga agacccagat 2040

gaaggcacag ctggagtgga gtccgctgtt ctgaggggat tcctcattct gggcaaagaa 2100

gacaggagat atgggccagc attaagcatc aatgaactga gcaaccttgc gaaaggagag 2160

aaggctaatg tgctaattgg gcaaggagac gtggtgttgg taatgaaacg aaaacgggac 2220

tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggatggc catcaattag 2280

<210> 4

<211> 2274

<212> DNA

<213> Artificial Sequence

<400> 4

atggatgtca atccgacctt acttttctta aaagtgccag cacaaaatgc tataagcaca 60

actttccctt ataccggaga ccctccttac agccatggga caggaacagg atacaccatg 120

gatactgtca acaggacaca tcagtactca gaaaagggaa gatggacaac aaacaccgaa 180

actggagcac cgcaactcaa cccgattgat gggccactgc cagaagacaa tgaaccaagt 240

ggttatgccc aaacagattg tgtattggaa gcaatggctt tccttgagga atcccatcct 300

ggtatttttg aaaactcgtg tattgaaacg atggaggttg ttcagcaaac acgagtagac 360

aagctgacac aaggccgaca gacctatgac tggactttaa atagaaacca gcctgctgca 420

acagcattgg ccaacacaat agaagtgttc agatcaaatg gcctcacggc caatgagtca 480

ggaaggctca tagacttcct taaggatgta atggagtcaa tgaaaaaaga agaaatgggg 540

atcacaactc attttcagag aaagagacgg gtgagagaca atatgactaa gaaaatgata 600

acacagagaa caataggtaa aaggaaacag agattgaaca aaaggggtta tctaattaga 660

gcattgaccc tgaacacaat gaccaaagat gctgagagag ggaagctaaa acggagagca 720

attgcaaccc cagggatgca aataaggggg tttgtatact ttgttgagac actggcaagg 780

agtatatgtg agaaacttga acaatcaggg ttgccagttg gaggcaatga gaagaaagca 840

aagttggcaa atgttgtaag gaagatgatg accaattctc aggacaccga actttctttc 900

accatcactg gagataacac caaatggaac gaaaatcaga atcctcggat gtttttggcc 960

atgatcacat atatgaccag aaatcagccc gaatggttca gaaatgttct aagtattgct 1020

ccaataatgt tctcaaacaa aatggcgaga ctgggaaaag ggtatatgtt tgagagcaag 1080

agtatgaaac ttagaactca aatacctgca gaaatgctag caagcattga tttgaaatat 1140

ttcaatgatt caacaagaaa gaagattgaa aaaatccgac cgctcttaat agaggggact 1200

gcatcattga gccctggaat gatgatgggc atgttcaata tgttaagcac tgtattaggc 1260

gtctccatcc tgaatcttgg acaaaagaga tacaccaaga ctacttactg gtgggatggt 1320

cttcaatcct ctgacgattt tgctctgatt gtgaatgcac ccaatcatga agggattcaa 1380

gccggagtcg acaggtttta tcgaacctgt aagctacttg gaatcaatat gagcaagaaa 1440

aagtcttaca taaacagaac aggtacattt gaattcacaa gttttttcta tcgttatgggg 1500

tttgttgcca atttcagcat ggagcttccc agttttgggg tgtctgggat caacgagtca 1560

gcggacatga gtattggagt tactgtcatc aaaaacaata tgataaacaa tgatcttggt 1620

ccagcaacag ctcaaatggc ccttcagttg ttcatcaaag attacaggta cacgtaccga 1680

tgccatagag gtgacacaca aatacaaacc cgaagatcat ttgaaataaa gaaactgtgg 1740

gagcaaaccc gttccaaagc tggactgctg gtctccgacg gaggcccaaa tttatacaac 1800

attagaaatc tccacattcc tgaagtctgc ctaaaatggg aattgatgga tgaggattac 1860

caggggcgtt tatgcaaccc actgaaccca tttgtcagcc ataaagaaat tgaatcaatg 1920

aacaatgcag tgatgatgcc agcacatggt ccagccaaaa acatggagta tgatgctgtt 1980

gcaacaacac actcctggat ccccaaaaga aatcgatcca tcttgaatac aagtcaaaga 2040

ggagtacttg aagatgaaca aatgtaccaa aggtgctgca atttatttga aaaattcttc 2100

cccagcagtt catacagaag accagtcggg atatccagta tggtggaggc tatggtttcc 2160

agagcccgaa ttgatgcacg gattgatttc gaatctggaa ggataaagaa agaagagttc 2220

actgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa atag 2274

<210> 5

<211> 2151

<212> DNA

<213> Artificial Sequence

<400> 5

atggaagatt ttgtgcgaca atgcttcaat ccgatgattg tcgagcttgc ggaaaaaaca 60

atgaaagagt atggggagga cctgaaaatc gaaacaaaca aatttgcagc aatatgcact 120

cacttggaag tatgcttcat gtattcagat ttccacttca tcaatgagca aggcgagtca 180

ataatcgtag aacttggtga tcctaatgca cttttgaagc acagatttga aataatcgag 240

ggaagagatc gcacaatggc ctggacagta gtaaacagta tttgcaacac tacaggggct 300

gagaaaccaa agtttctacc agatttgtat gattacaagg aaaatagatt catcgaaatt 360

ggagtaacaa ggagagaagt tcacatatac tatctggaaa aggccaataa aattaaatct 420

gagaaaacac acatccacat tttctcgttc actggggaag aaatggccac aagggccgac 480

tacactctcg atgaagaaag cagggctagg atcaaaacca ggctattcac cataagacaa 540

gaaatggcca gcagaggcct ctgggattcc tttcgtcagt ccgagagagg agaagagaca 600

attgaagaaa ggtttgaaat cacaggaaca atgcgcaagc ttgccgacca aagtctcccg 660

ccgaacttct ccagccttga aaattttaga gcctatgtgg atggattcga accgaacggc 720

tacattgagg gcaagctgtc tcaaatgtcc aaagaagtaa atgctagaat tgaacctttt 780

ttgaaaacaa caccacgacc acttagactt ccgaatgggc ctccctgttc tcagcggtcc 840

aaattcctgc tgatggatgc cttaaaatta agcattgagg acccaagtca tgaaggagag 900

ggaataccgc tatatgatgc aatcaaatgc atgagaacat tctttggatg gaaggaaccc 960

aatgttgtta aaccacacga aaagggaata aatccaaatt atcttctgtc atggaagcaa 1020

gtactggcag aactgcagga cattgagaat gaggagaaaa ttccaaagac taaaaatatg 1080

aaaaaaacaa gtcagctaaa gtgggcactt ggtgagaaca tggcaccaga aaaggtagac 1140

tttgacgact gtaaagatgt aggtgatttg aagcaatatg atagtgatga accagaattg 1200

aggtcgcttg caagttggat tcagaatgag ttcaacaagg catgcgaact gacagattca 1260

agctggatag agcttgatga gattggagaa gatgtggctc caattgaaca cattgcaagc 1320

atgagaagga attatttcac atcagaggtg tctcactgca gagccacaga atacataatg 1380

aagggggtgt acatcaatac tgccttactt aatgcatctt gtgcagcaat ggatgatttc 1440

caattaattc caatgataag caagtgtaga actaaggagg gaaggcgaaa gaccaacttg 1500

tatggtttca tcataaaagg aagatcccac ttaaggaatg acaccgacgt ggtaaacttt 1560

gtgagcatgg agttttctct cactgaccca agacttgaac cacacaaatg ggagaagtac 1620

tgtgttcttg agataggaga tatgcttcta agaagtgcca taggccaggt ttcaaggccc 1680

atgttcttgt atgtgaggac aaatggaacc tcaaaaatta aaatgaaatg gggaatggag 1740

atgaggcgtt gtctcctcca gtcacttcaa caaattgaga gtatgattga agctgagtcc 1800

tctgtcaaag agaaagacat gaccaaagag ttctttgaga acaaatcaga aacatggccc 1860

attggagagt ctcccaaagg agtggaggaa agttccattg ggaaggtctg caggacttta 1920

ttagcaaagt cggtatttaa cagcttgtat gcatctccac aactagaagg attttcagct 1980

gaatcaagaa aactgcttct tatcgttcag gctcttaggg acaatctgga acctgggacc 2040

tttgatcttg gggggctata tgaagcaatt gaggagtgcc taattaatga tccctgggtt 2100

ttgcttaatg cttcttggtt caactccttc cttacacatg cattgagtta g 2151

<210> 6

<211> 1497

<212> DNA

<213> Artificial Sequence

<400> 6

atggcgtccc aaggcaccaa acggtcttac gaacagatgg agactgatgg agaacgccag 60

aatgccactg aaatcagagc atccgtcgga aaaatgattg gtggaattgg acgattctac 120

atccaaatgt gcacagaact taaactcagt gattatgagg gacggttgat ccaaaacagc 180

ttaacaatag agagaatggt gctctctgct tttgacgaaa ggagaaataa atacctggaa 240

gaacatccca gtgcgggggaa agatcctaag aaaactggag gacctatata cagaagagta 300

aacggaaagt ggatgagaga actcatcctt tatgacaaag aagaaataag gcgaatctgg 360

cgccaagcta ataatggtga cgatgcaacg gctggtctga ctcacatgat gatctggcat 420

tccaatttga atgatgcaac ttatcagagg acaagggctc ttgttcgcac cggaatggat 480

cccaggatgt gctctctgat gcaaggttca actctcccta ggaggtctgg agccgcaggt 540

gctgcagtca aaggagttgg aacaatggtg atggaattgg tcaggatgat caaacgtggg 600

atcaatgatc ggaacttctg gaggggtgag aatggacgaa aaacaagaat tgcttatgaa 660

agaatgtgca acattctcaa agggaaattt caaactgctg cacaaaaagc aatgatggat 720

caagtgagag agagccggaa cccagggaat gctgagttcg aagatctcac ttttctagca 780

cggtctgcac tcatattgag agggtcggtt gctcacaagt cctgcctgcc tgcctgtgtg 840

tatggacctg ccgtagccag tgggtacgac tttgaaagag agggatactc tctagtcgga 900

atagaccctt tcagactgct tcaaaacagc caagtgtaca gcctaatcag accaaatgag 960

aatccagcac acaagagtca actggtgtgg atggcatgcc attctgccgc atttgaagat 1020

ctaagagtat tgagcttcat caaagggacg aaggtggtcc caagagggaa gctttccact 1080

agaggagttc aaattgcttc caatgaaaat atggagacta tggaatcaag tacacttgaa 1140

ctgagaagca ggtactgggc cataaggacc agaagtggag gaaacaccaa tcaacagagg 1200

gcatctgcgg gccaaatcag catacaacct acgttctcag tacagagaaa tctccctttt 1260

gacagaacaa ccgttatggc agcattcact gggaatacag aggggagaac atctgacatg 1320

aggaccgaaa tcataaggat gatggaaagt gcaagaccag aagatgtgtc tttccagggg 1380

cggggagtct tcgagctctc ggacgaaaag gcagcgagcc cgatcgtgcc ttcctttgac 1440

atgagtaatg aaggatctta tttcttcgga gacaatgcag aggagtacga caattaa 1497

<210> 7

<211> 982

<212> DNA

<213> Artificial Sequence

<400> 7

atgagtcttc taaccgaggt cgaaacgtac gttctctcta tcatcccgtc aggccccctc 60

aaagccgaga tcgcacagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120

gttctcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa ggggatttta 180

ggatttgtgt tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240

caaaatgccc ttaatgggaa cggggatcca aataacatgg acaaagcagt taaactgtat 300

aggaagctca agagggagat aacattccat ggggccaaag aaatctcact cagttattct 360

gctggtgcac ttgccagttg tatgggcctc atatacaaca ggatgggggc tgtgaccact 420

gaagtggcat ttggcctggt atgtgcaacc tgtgaacaga ttgctgactc ccagcatcgg 480

tctcataggc aaatggtgac aacaaccaac ccactaatca gacatgagaa cagaatggtt 540

ttagccagca ctacagctaa ggctatggag caaatggctg gatcgagtga gcaagcagca 600

gaggccatgg aggttgctag tcaggctagg caaatggtgc aagcgatgag aaccattggg 660

actcatccta gctccagtgc tggtctgaaa aatgatcttc ttgaaaattt gcaggcctat 720

cagaaacgaa tgggggtgca gatgcaacgg ttcaagtgat cctctcgcta ttgccgcaaa 780

tatcattggg atcttgcact tgatattgtg gattcttgat cgtctttttt tcaaatgcat 840

ttaccgtcgc tttaaatacg gactgaaagg agggccttct acggaaggag tgccaaagtc 900

tatgagggaa gaatatcgaa aggaacagca gagtgctgtg gatgctgacg atggtcattt 960

tgtcagcata gagctggagt aa 982

<210> 8

<211> 838

<212> DNA

<213> Artificial Sequence

<400> 8

atggatccaa acactgtgtc aagctttcag gtagattgct ttctttggca tgtccgcaaa 60

cgagttgtag accaagaact aggtgatgcc ccattccttg atcggcttcg ccgagatcag 120

aaatccctaa gaggaagggg cagcactctt ggtctggaca tcgagacagc cacacgtgct 180

ggaaagcaga tagtggagcg gattctgaaa gaagaatccg atgaggcact taaaatgacc 240

atggcctctg tacctgcgtc gcgttaccta accgacatga ctcttgagga aatgtcaagg 300

gaatggtcca tgctcatacc caagcagaaa gtggcaggcc ctctttgtat cagaatggac 360

caggcgatca tggataaaaa catcatactg aaagcgaact tcagtgtgat ttttgaccgg 420

ctggagactc taatattgct aagggctttc accgaagagg gagcaattgt tggcgaaatt 480

tcaccattgc cttctcttcc aggacatact gctgaggatg tcaaaaatgc agttggagtc 540

ctcatcggag gacttgaatg gaatgataac acagttcgag tctctgaaac tctacagaga 600

ttcgcttgga gaagcagtaa tgagaatggg agacctccac tcactccaaa acagaaacga 660

gaaatggcgg gaacaattag gtcagaagtt tgaagaaata agatggttga ttgaagaagt 720

gagacacaaa ctgaaggtaa cagagaatag ttttgagcaa ataacattta tgcaagcctt 780

acatctattg cttgaagtgg agcaagagat aagaactttc tcatttcagc ttatttaa 838

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 9

atggagaaca tagtacttct tcttg 25

<210> 10

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 10

gccccgaaca ggcctctagt ttctcttaga ggactatttc tgagc 45

<210> 11

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 11

actagaggcc tgttcggggc gatagcaggg t 31

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 12

ttaaatgcaa attctgcact gtaac 25

Claims (6)

1. The artificially recombined H5N8 influenza virus strain H5-Re14 is preserved in China center for type culture Collection, is addressed to Wuhan university in Wuhan, China, and has the preservation number of CCTCC NO: v202160, preservation time 2021, 8 months and 3 days.

2. The strain H5-Re14 as claimed in claim 1, for use in the preparation of a medicament for the prevention of diseases caused by the H5N8 influenza virus.

3. The use of claim 2, wherein the influenza virus H5N8 is the A/swan/Shanxi/4-1/2020(H5N8) strain.

4. A vaccine comprising the strain H5-Re14 of claim 1.

5. The strain H5-Re14 of claim 1, wherein the strain H5-Re14 comprises:

(1) designing HA gene specific amplification and mutation primers, amplifying HA1 and HA2 segments of HA genes of highly pathogenic H5N8 subtype avian influenza virus A/swan/Shanxi/4-1/2020(H5N8) strains by using an RT-PCR method, and mutating amino acid at a cracking site of the HA genes from-REKRRKR-to-RETR-while amplifying the HA1 segments, so that the molecular characteristics of the highly pathogenic avian influenza virus are mutated into the molecular characteristics of a typical low pathogenic avian influenza virus; then, an HA segment with a cracking site being the molecular characteristic of the typical low-pathogenicity avian influenza virus is amplified by utilizing an overlap extension reaction, and a pBD plasmid is inserted to construct a recombinant HA gene bidirectional transcription vector;

(2) amplifying the NA gene full-length segment of H5N8 subtype avian influenza virus A/swan/Shanxi/4-1/2020(H5N8) by using an RT-PCR method, inserting a pBD plasmid, and constructing a recombinant NA gene bidirectional transcription vector;

(3) mixing 2 recombinant pBD bidirectional transcription vector plasmids of HA and NA genes constructed in the steps (1) and (2) with 6 pBD bidirectional transcription vector plasmids of PB2, PB1, PA, NP, M and NS genes of an influenza virus chick embryo high-titer adaptive strain A/PR/8/34(H1N1), inoculating 293T cells with a transfection reagent, and inoculating the cells and supernatant harvested after culture into a chick embryo to obtain an H5N8 avian influenza recombinant virus strain H5-Re14 with HA activity.

6. The preparation method of claim 5, wherein the primer pair for amplifying the HA1 gene is shown as SEQ ID No.9 and SEQ ID No.10, wherein SEQ ID No.10 is a mutation primer of HA cleavage site; the primer pair for amplifying the HA2 gene is shown as SEQ ID No.11 and SEQ ID No. 12.

Images