Influenza HA protein recombinant herpesvirus turkey strain and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses an influenza HA protein recombinant turkey herpesvirus strain, the preservation number of the recombinant turkey herpesvirus strain is CGMCC No.46642, and the recombinant turkey herpesvirus strain is preserved in China General Microbiological Culture Collection Center; the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is July 23, 2025. The recombinant turkey herpesvirus has the characteristics that: 1, the recombinant turkey herpesvirus has good safety and immunogenicity, can induce strong body fluid and cellular immune response, can realize early immunization through intraembryonic or 1-day-old inoculation, and reduces interference of maternal antibodies; and 2, the HA gene of the H7 subtype AIV and the virus HA gene of the H5 subtype AIV are inserted into the virus strain, the two genes are independently screened by the applicant, and after being loaded on an HVT vector, the HA gene and the virus HA gene can show relatively comprehensive protection aiming at the corresponding subtype. Meanwhile, the invention also provides application and a vaccine of the recombinant herpesvirus of turkeys.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a recombinant turkey herpesvirus strain containing influenza HA protein and its uses. Background Technology

[0002] The preparation of recombinant viruses using HVT (turkey herpesvirus) as a basic vector is a topic of extensive research by scholars in various countries. The applicant has conducted relatively in-depth research based on HVT, as evidenced by the applicant's prior applications:

[0003] Patent application CN118879643A, entitled "Recombinant Turkey Herpesvirus HVT-H9-IBD Expressing Two Exogenous Genes and Its Application," discloses the following: Target gene 1 and target gene 2 are inserted between the HVT053 and HVT054 sites and between the HVT065 and HVT066 sites in the UL region of the turkey herpesvirus; or, target gene 1 and target gene 2 are inserted between the HVT053 and HVT054 sites and between the HVT087 and HVT088 sites in the UL region of the turkey herpesvirus. Target gene 1 is an HA gene with a CMV promoter and a bgH terminator, and target gene 2 is a VP2 gene with an mCMV promoter and an SV40 terminator. Recombinant virus 1 and recombinant virus 2 are obtained respectively.

[0004] This application is based on further research on HVT-loaded HA genes and has yielded some new research results, which leads to this application.

[0005] For further research on existing technologies related to HVT-loaded HA genes, the following technical literature can be consulted:

[0006] The patent application with publication number CN 114828882 A is for a multivalent HVT vector vaccine.

[0007] The patent application with publication number CN 108543067 A is for a recombinant turkey herpesvirus vaccine strain that co-expresses H5 subtype avian influenza HA protein and infectious bursal disease VP2 protein.

[0008] The patent application with publication number MX2024003750A0 is entitled "A novel recombinant turkey herpesvirus expressing avian influenza virus (AIV) H5 hemagglutinin protein, which can provide immunogenicity and protection to chickens, reduce virus shedding, and resist AIV H5N1 attack."

[0009] The announcement number is CN 110218706 B, and the subject is the construction and application of recombinant turkey herpesvirus expressing the HA protein of H7N9 subtype highly pathogenic avian influenza virus.

[0010] Through the above literature review, we can find that existing research on the HA gene loaded onto HVT vectors is relatively abundant. However, most existing studies are limited to the insertion site, leaving gaps in understanding the relevant performance of the HA gene and its cross-protective performance against the same viral subtype after being loaded onto the HVT vector. Summary of the Invention

[0011] The purpose of this invention is to provide a recombinant turkey herpesvirus strain containing the influenza HA protein. This recombinant turkey herpesvirus is characterized by: 1. Good safety and immunogenicity, capable of inducing strong humoral and cellular immune responses, and early immunization through intraembryonic inoculation, or early immunization through inoculation at 1 day of age, reducing interference from maternal antibodies; 2. This strain incorporates the HA gene of H7 subtype AIV and the virulence HA gene of H5 subtype AIV. Both genes were independently screened by the applicant and, after verification, exhibit comprehensive protection against their respective subtypes when loaded onto the HVT vector.

[0012] In addition, the present invention also provides the use of the recombinant turkey herpesvirus and a vaccine.

[0013] To achieve the above objectives, the present invention provides the following technical solution:

[0014] A recombinant turkey herpesvirus strain with influenza HA protein, the accession number of which is CGMCC No. 46642, is deposited at the China General Microbiological Culture Collection Center; the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is July 23, 2025, and the classification name is: Turkey Herpesvirus.

[0015] In addition, the present invention also discloses the use of the influenza HA protein recombinant turkey herpesvirus strain described above in the preparation of a vaccine.

[0016] In the above-described uses, the vaccine is a vaccine for the prevention of Marek's disease.

[0017] In the above-described uses, the vaccine is a vaccine for preventing infection with H7 subtype AIV virus.

[0018] In the above-mentioned uses, the vaccine is a vaccine for preventing infection with H5 subtype AIV virus.

[0019] Furthermore, this invention also discloses the use of the influenza HA protein recombinant turkey herpesvirus strain described above as a novel viral vector.

[0020] Finally, the present invention also discloses a vaccine containing a recombinant turkey herpesvirus strain containing the influenza HA protein as described above.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. This invention uses HVT-FC126 strain (GenBank: AF291866.1) as a viral vector and employs CRISPR / Cas9 and homologous end ligation (HDR) technology to insert the HA gene of CK / HB / 24 / 21 (H7N9) into the US region. Further, using rHVT-H7 virus as a vector, the H5 gene is inserted into the HVT UL region, successfully constructing the rHVT-H5-H7 recombinant virus. This recombinant virus stably expresses H5 and H7 proteins, with plaque size and proliferation level similar to the parental strain, and is stably passaged in vitro for at least 20 generations.

[0023] 2. This invention evaluated the immunoprotective effect of rHVT-H5-H7 recombinant virus against H5N1 and H7N9 subtype AIVs. Nasal challenge was performed on SPF chickens on day 35 post-immunization. 5.0 EID 50 The dosage of the virus was adjusted. When infected with homologous viruses CK / GX / 34 / 23 (H5N1) or CK / HB / 24 / 21 (H7N9), all immunized chickens survived for 14 days without viral shedding or organ replication, while all HVT-FC126-immunized control chickens died within 4–5 days after challenge with either virus. When infected with representative viruses from two different branches of H5, the survival rates of the immunized groups were 83.3% and 100%, respectively, with only 1 (1 / 6) and 0 (0 / 6) chickens showing viral shedding. When challenged with two heterologous H7N9 subtype viruses, all challenged chickens survived, with 1 (1 / 6) and 0 (0 / 6) chickens in each group showing viral shedding.

[0024] Two conclusions can be drawn from the above-mentioned experiments on immune protection:

[0025] The recombinant strain of this invention provides strong protection; the recombinant strain of this invention also exhibits strong cross-protection against viruses of the same subtype. Attached Figure Description

[0026] Figure 1A This is a phylogenetic tree of the HA gene in the H5 subtype AIV 2.3.4.4b branch;

[0027] Figure 1B Antigenic map of H5Ny virus;

[0028] Figure 2A This is a genetic evolution diagram of the HA gene of the H7 subtype influenza virus.

[0029] Figure 2B An antigenic map of the H7N9 virus;

[0030] Figure 3 Diagram illustrating the construction strategy for the recombinant virus rHVT-H5-H7;

[0031] Figure 4 Figure showing the identification results of H7 and H5 protein expression in the rHVT-H5-H7 recombinant virus;

[0032] Figure 5 The results of antibody titer detection in chickens immunized with rHVT-H5-H7 recombinant virus were obtained using HI assay.

[0033] Figure 6 Survival curve of immunized chickens after challenge with H5N1 subtype avian influenza virus;

[0034] Figure 7 Image showing the results of H5N1 AIV viral titer detection in the lungs on the third day after challenge;

[0035] Figure 8 Figure showing the evaluation results of challenge and cohabitation transmission of rHVT-H5-H7 immunized chickens with H5 subtype avian influenza virus;

[0036] Figure 9 A curve showing the survival rate of chickens immunized with H7N9 AIV.

[0037] Figure 10 Image showing the results of H7N9 AIV viral titer detection in the lungs on the third day after challenge;

[0038] Figure 11 The figure shows the evaluation results of challenge and cohabitation transmission of rHVT-H5-H7 avian influenza virus in chickens immunized with H7 subtype avian influenza. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1: Determination of the inserted gene in the HVT live vector vaccine

[0041] First, the prevalence characteristics of H5 and H7 subtypes of AIV in my country were studied.

[0042] Between 2021 and 2024, the applicant isolated a total of 15 strains of H5 subtype AIV (13 strains of H5N1 and 2 strains of H5N8) and 5 strains of H7N9 subtype AIV from 14 monitoring sites across the country. Phylogenetic analysis was performed using the gene sequences of AIVs in public databases, as shown in the attached figure. Figure 1A As shown, the HA genes of the isolated H5 subtype AIVs all belong to the 2.3.4.4b branch, which can be further divided into three sub-branches: Clade I, Clade II and Clade III.

[0043] The HA gene of the isolated H7 subtype AIV is similar in evolutionary relationship to previous H7N9 isolates from the Yangtze River Delta region of my country, as shown in the attached figure. Figure 2A As shown, it is also divided into three sub-branches: Clade I, Clade II, and Clade III.

[0044] Analysis of antigenic variation of H5 subtype avian influenza virus:

[0045] To understand the correlation between the current strain and the antigenicity of the commercially available vaccine strain H5 Re-14, this invention selected three representative strains from different regions and time periods for gene homology analysis. These included Clade I branch strain ENV / LN / 4 / 23, Clade II branch strain CK / GX / 34 / 23, and Clade III branch strain LB / ZJ / 1 / 21 as representative strains of each branch to prepare antiserum. The cross-reactivity of the prepared antiserum to these viruses was evaluated by hemagglutinin inhibition assay (HI).

[0046] As shown in Table 1, the antibody titer of H5-Re14 antiserum against the homologous virus was 256. Of the seven viruses tested, four showed good reactivity with the H5-Re14 antiserum, with HI titers equal to or two-fold lower than those of antiserums prepared from the same branch of the virus. Two other viruses showed HI titers eight-fold lower than those of antiserums prepared from the same branch of the virus. LB / ZJ / 1 / 21 showed the worst reactivity with H5-Re14, differing by a factor of 32. These results indicate that most H5Ny virus strains in this study showed good cross-reactivity with the antiserum induced by the H5-Re14 vaccine, but some viruses also showed reduced reactivity with the H5-Re14 antiserum.

[0047] Table 1. Antigenicity analysis of different branches of the H5 circulating strains.

[0048]

[0049]

[0050] As shown in Table 2, compared to the antiserum prepared from the H5-Re14 strain, the antiserum of CK / GX / 34 / 23 showed better cross-reactivity with other strains. In particular, the antiserum for ENV / LN / 4 / 23 virus had an HI titer 8 times lower than its homologous titer (32 times lower than that of H5-Re14). When the antiserum of the Clade II strain clade strain CK / GX / 34 / 23 reacted with the Clade I strain, the HI titer differed by a factor of 2, and the Clade II strain had a higher isolation rate among all strains. For LB / ZJ / 1 / 21, the clade containing this strain had a relatively small proportion, and no strains from this clade were observed after 2021. Furthermore, this strain could be classified into a new antigenic group (…). Figure 1B ); Figure 1B The antigenic profile shown is based on the HI test data in Table 1. Each coordinate unit in the figure represents a 2-fold difference in HI titer, i.e., an antigenic distance. The boxes represent antiserum produced from the H5-Re14 vaccine strain. The ellipses represent the detection virus used for antigen analysis.

[0051] Analysis of antigenic variation of H7 subtype avian influenza virus

[0052] Representative strains from different branches were selected to prepare antisera to analyze changes in H7 antigenicity. As shown in Table 2, the antibody titer of the H7-Re4 antiserum against the homologous virus was 512. Two viruses reacted well with the H7-Re4 antiserum, with HI titers 2 to 4 times lower than those of antisera from the same branch. Two other viruses showed HI titers 16-32 times lower than those of antisera from the same branch. CK / HB / 27 / 21 showed the worst reaction with H7-Re4, differing by 64 times. These results indicate that most H7N9 virus strains in this study still exhibit some degree of cross-reactivity with the antiserum induced by the H7-Re4 vaccine, but a decrease in reactivity with the H7-Re4 antiserum was also observed in some viruses.

[0053] In addition, according to Figure 2B The antigenicity of the isolated strains in this study is correlated with the branching. The antiserum of CK / HB / 24 / 21 showed relatively good reactivity with other strains, and this branch of strains is currently prevalent, with the highest isolation rate in the past two years. Therefore, CK / HB / 24 / 21 can be selected as a candidate H7 strain for the preparation of HVT live vector vaccine.

[0054] Table 2. Antigenicity analysis of H7 epidemic strains from different subclades.

[0055]

[0056]

[0057] Figure 2BThe antigenic profile shown is based on hemagglutination inhibition (HI) test data from Table 2-9. Each coordinate unit in the graph represents a 2-fold difference in HI titer, i.e., antigenic distance. Boxes represent antiserum produced from the H7-Re4 vaccine strain. Circles represent the detection virus used for antigen analysis.

[0058] Based on the isolation rate and antigenicity of different branch viruses, this invention identified the HA gene of the CK / GX / 34 / 23 (H5N1) and CK / HB / 24 / 21 (H7N9) strains as the insertion gene for constructing H5 and H7 recombinant HVT live vector vaccines.

[0059] The sequence of the HA gene (with the multibasic cleavage site removed) of the above CK / GX / 34 / 23(H5N1) is shown in SEQ ID NO.1;

[0060] The sequence of the HA gene (with the multibasic cleavage site removed) of the above CK / HB / 24 / 21(H7N9) is shown in SEQ ID NO.2.

[0061] Example 2: Construction of recombinant turkey herpesvirus rHVT-H7

[0062] 1. Construction of the virus strain:

[0063] In this invention, the HA gene expression cassette containing the H7 subtype AIV is inserted into the HVT genome. The insertion region is a non-essential region for viral growth in the US region of the HVT (GenBank: AF291866.1) genome.

[0064] To achieve the above objectives, this invention utilizes CRISPR / Cas9 and homologous end ligation (HDR) technology for virus rescue. First, a recombinant plasmid for homologous recombination was constructed. This plasmid contains a homologous sequence upstream of the insertion site, a promoter gene, the HA gene of the H7 subtype AIV, a poly(A) tail, a GFP gene expression nucleus, and a homologous sequence downstream of the insertion site. This recombinant plasmid was co-transfected with a CRISPR / Cas9 plasmid into CEF cells. After the appearance of green fluorescent plaques, one plaque was picked and serially diluted 3-fold, then transferred to three individual wells of a 6-well plate. As new fluorescent plaques appeared, plaque purification was continued using limiting dilution until all plaques in the wells were fluorescent. After virus identification and purification by PCR, the GFP expression cassette was deleted using the Cre enzyme system until all plaques in the wells were non-fluorescent. Virus identification and purification were then performed again by PCR. This non-fluorescent virus was named a recombinant turkey herpesvirus rHVT-H7 strain, which provides good protection against H7N9 infection.

[0065] Subsequently, using rHVT-H7 as a vector platform, the HA gene of the H5 subtype AIV was inserted to construct the recombinant virus rHVT-H5-H7. A schematic diagram of the DNA sequence of the above recombinant virus is attached. Figure 3 As shown. The specific solution is as follows:

[0066] In this invention, the HA gene expression cassette containing the H5 subtype AIV is inserted into the rHVT-H7 genome. The insertion region is a non-essential region for viral growth in the UL region of the HVT (GenBank:AF291866.1) genome.

[0067] To achieve the above objectives, this invention first constructed a recombinant plasmid for homologous recombination. This recombinant plasmid contains a homologous sequence upstream of the insertion site, a promoter gene, the HA gene of H5 subtype AIV, a poly(A) tail, a GFP gene expression nucleus, and a homologous sequence downstream of the insertion site. Using rHVT-H7 as a vector platform, and referring to the method similar to that in specific implementation (1), a non-fluorescent virus carrying the HA gene of H5 subtype AIV and the HA gene of H7 subtype AIV was successfully rescued and named a recombinant turkey herpesvirus rHVT-H5-H7 strain. In addition to providing protection against H7N9 infection, it also provides good protection against H5N1 infection. This virus strain was deposited on July 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46642 and classification name: Turkey Herpesvirus.

[0068] Example 3: Identification of H7 and H5 protein expression in the rHVT-H5-H7 recombinant virus

[0069] CEF cells were infected with 100 PFU of rHVT-H5-H7 recombinant virus and HVT-FC126 parental virus, respectively. After 72 h, the cells were fixed and HA protein expression was detected by IFA. (See attached image) Figure 4 As shown, in the rHVT-H5-H7 infected group, both H7 HA protein (green fluorescence) and H5 HA protein (red fluorescence) signals were observed in the viral plaque region, indicating that this recombinant virus can stably co-express both HA antigens in CEF cells. In contrast, only the green fluorescence signal of H7 HA was detected in rHVT-H7, while no specific fluorescence signal was observed in the HVT-FC126 group. These results further verify the successful expression of dual HA genes in the rHVT-H5-H7 recombinant virus.

[0070] Example 4: Evaluation of Immunization Effect

[0071] To evaluate the safety of the rHVT-H5-H7 recombinant virus and the humoral immune response it induces, this invention immunized 10 one-day-old SPF chickens by subcutaneous injection in the neck with a dose of 3000 PFU.

[0072] No obvious clinical symptoms were observed in the experimental chickens within 5 weeks after immunization, indicating that the recombinant virus has good biosafety.

[0073] As attached Figure 5 As shown, specific HI antibodies for H5 and H7 subtypes can be detected from the second week after immunization, and the antibody titers gradually increase over time. By the fifth week, the H5 antibody titer is between 3 and 7 log2, with an average titer of approximately 4 log2; the H7 antibody titer is between 4 and 8 log2, with an average titer of approximately 5 log2.

[0074] The results showed that the rHVT-H5-H7 recombinant virus had good immunogenicity and could induce the production of specific antibodies against both H5 and H7 subtypes.

[0075] Example 5

[0076] (1) Evaluation of the immunoprotective efficacy of rHVT-H5-H7 live vaccine against H5 subtype AIV in chickens

[0077] As attached Figure 6 As shown, 6 chickens in the rHVT-H5-H7 immunized group were infected for 10... 5.0 EID 50 After being given the same strain CK / GX / 34 / 23 (Clade II), all chickens survived the 14-day observation period (6 / 6).

[0078] 10 nasal cavity virus attack 5.0 EID 50 After administration of heterologous strains ENV / LN / 4 / 23 (Clade I) and LB / ZJ / 1 / 21 (Clade III) (both H5 subtype strains), the survival rates of immunized chickens were 100% (6 / 6) and 83.3% (5 / 6), respectively. In contrast, in the control group (immunized with HVT-FC126), all chickens died within 4 days after challenge (0 / 6) after infection with either strain, indicating that the rHVT-H5-H7 vaccine has good protective ability against H5N1 virus and good cross-protection effect.

[0079] On the third day after viral challenge, three chickens were randomly selected from each of the vaccine-immunized groups for euthanasia and lung tissue was collected; if the chickens in the control group had died, lung tissue was separated from the dead chickens for testing.

[0080] As attached Figure 7As shown, in control chickens (immunized with HVT-FC126), significant viral replication was detected in lung tissue after inoculation with the CK / GX / 34 / 23 (Clade II) strain, with viral titers ranging from 10. 5.75 ~10 7.25 EID 50 / mL, with an average titer of 10 6.25 EID 50 / mL, and significant viral replication was also observed in other H5N1 subtype virus challenge groups. In contrast, no viral replication was detected in the lung tissue of immunized chickens, suggesting that the rHVT-H5-H7 vaccine can effectively inhibit viral replication in the lungs.

[0081] Figure 7 The viral titer in lung tissue from necropsy-dissected chickens in each group was measured 3 days post-infection. Three chickens were in each group, and each sample had two replicates. Differences between the immunized challenge groups and the challenge control group were analyzed using a two-tailed t-test; ***, p < 0.001. 0.75 EID 50 / mL is the detection limit;

[0082] To assess the vaccine's ability to block virus transmission, on the second day after challenge, six immunized chickens (rHVT-H5-H7 immunized group) were added to each vaccine group as cohabiting chickens to monitor virus transmission. (See attached image) Figure 8 As shown, oral and anal swabs were collected from challenged chickens on days 3 and 5 (DPI) and from cohabiting chickens on days 3 and 5 (DPC) for qPCR testing after inoculation with the homologous strain CK / GX / 34 / 23 (Clade II). No virus was detected in any of the swab samples.

[0083] Figure 8 In this study, oral and anal swabs were collected from challenged and cohabiting chickens at DPI (days 3 and 5) and DPC (days 3 and 5), respectively, and viral shedding was detected by real-time quantitative PCR. 0.75 EID 50 / mL is the detection limit.

[0084] One chicken died after being inoculated with the heterologous strain LB / ZJ / 1 / 21 (Clade III). Virus titers of 10 were detected in oral swabs collected on days 3 and 5 prior to death. 3.5 EID 50 / mL, and no virus was detected in any of the swab samples from the other challenged chickens and all cohabiting chickens. After challenged with another heterologous strain ENV / LN / 4 / 23 (Clade I), no virus was detected in any of the swab samples from the challenged chickens and all cohabiting chickens, indicating that the rHVT-H5-H7 vaccine has a good transmission blocking effect.

[0085] Example 6: Evaluation of the immunoprotective efficacy of rHVT-H5-H7 live vaccine against H7 subtype AIV in chickens.

[0086] In evaluating the immunoprotective efficacy against H7 subtype avian influenza virus, in addition to using the homologous strain CK / HB / 24 / 21 (Clade I), heterologous strains CK / SD / F0303 / 22 (Clade II) and CK / HB / 27 / 21 (Clade III), which have significant antigenic differences from the currently commercialized vaccine strain H7-Re14, were also selected for challenge experiments. The challenge dose for both strains was 10. 5 EID 50 / Only.

[0087] As attached Figure 9 As shown, all chickens in the vaccine group survived within 14 days after challenge, demonstrating good cross-protection. In contrast, the HVT-FC126 immunized control group survived for 10 days after infection. 5 EID 50 After being infected with the H7N9 virus, all chickens died within 5 days of being challenged.

[0088] On the third day after viral infection, three animals were randomly selected from both the vaccine group and the control group for euthanasia, and lung tissue was collected. Figure 10 As shown, significant viral replication was detected in the lung tissue of control group chickens. After challenge with CK / HB / 24 / 21 (Clade I), the viral titer in the lung tissue was 10. 6.25 ~10 8.25 EID 50 The average titer is 10. 7.25 EID 50 Significant viral replication was detected in all other groups. No virus was detected in the lungs of the immunized chickens.

[0089] Figure 10 The viral titer in lung tissue from necropsy-dissected chickens in each group was measured 3 days post-infection. Three chickens were in each group, and each sample had two replicates. Differences between the immunized challenge groups and the challenge control group were analyzed using a two-tailed t-test; ***, p < 0.001. 0.75 EID 50 / mL is the detection limit.

[0090] To evaluate the immunoprotective effect of the rHVT-H5-H7 vaccine against H7 subtype avian influenza virus, this embodiment refers to the method of H5 subtype challenge experiment. Oral and cloacal swabs were collected on days 3 and 5 post-challenge (DPI), and on days 3 and 5 post-challenge (DPC) in cohabiting chickens, for virus detection. (See attached...) Figure 11As shown, after infection with the homologous strain CK / HB / 24 / 21 (Clade I), no virus was detected in any samples, indicating that the vaccine can effectively block viral infection and its transmission. However, when infected with the heterologous strain CK / SD / F0303 / 22 (Clade II), the virus was detected in oral swabs on days 3 and 5 post-challenge, and in anal swabs on day 3. The virus was not detected in any swabs from the remaining chickens or any roosting chickens. When infected with another heterologous strain CK / HE / F0530 / 23 (Clade III), no virus was detected in any swab samples from the challenged chickens or roosting chickens, indicating that the vaccine also has good transmission blocking ability against this strain.

[0091] Figure 11 Oral and anal swabs were collected from challenged and cohabiting chickens at DPI (days 3 and 5) and DPC (days 3 and 5), respectively, to detect viral shedding using real-time quantitative PCR. 0.75 EID 50 / mL is the detection limit.

[0092] Based on the above experiments, we can draw the following conclusions:

[0093] 1. By analyzing the antigenic variations of H5 and H7 subtype avian influenza viruses, it can be theoretically proven that the HA gene selected in this invention has strong cross-protection against the same subtype; this protection was verified in Examples 5 and 6.

[0094] 2. Through Examples 4 to 6, we can see that the recombinant strain of the present invention has strong protective effect.

Claims

1. A recombinant turkey herpesvirus strain containing influenza HA protein, characterized in that, The recombinant turkey herpesvirus has the accession number CGMCC No. 46642 and is deposited at the China General Microbiological Culture Collection Center; the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is July 23, 2025, and the classification name is: Turkey Herpesvirus.

2. Use of the influenza HA protein recombinant turkey herpesvirus strain as described in claim 1 to prepare a vaccine.

3. The use according to claim 2, characterized in that, The vaccine in question is for the prevention of Marek's disease.

4. The use according to claim 2, characterized in that, The vaccine in question is for the prevention of infection with the H7 subtype AIV virus.

5. The use according to claim 2, characterized in that, The vaccine in question is for the prevention of infection with the H5 subtype AIV virus.

6. Use of the influenza HA protein recombinant turkey herpesvirus strain as described in claim 1 as a novel viral vector.

7. A vaccine, characterized in that, The recombinant turkey herpesvirus strain containing the influenza HA protein as described in claim 1.

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