Newcastle disease virus vector vaccine strains expressing type C aMPV F and G proteins and their application

Abstract

The application provides a newcastle disease virus vector vaccine strain expressing type C aMPV F protein and G protein and an application thereof, and belongs to the technical field of veterinary vaccine strains.The NDV vector vaccine strain constructed by the application is used for preventing and controlling type C avian pneumovirus of waterfowls, and solves the current market dilemma that there is no aMPV vaccine.Newcastle disease virus as a host vector is genetically modified, the virulence is attenuated, the safety is good, the duck has no pathogenicity, and the vector vaccine solves the safety risk that the aMPV live attenuated vaccine is prone to virulence reversion in the field;the vector vaccine has good immunization effect, can induce the body fluid immunity of the body, can induce the cellular immunity and mucosal immunity of the body, protects the immunized animals in all directions, can provide complete protection for aMPV-C wild strains and NDV-VII wild strains, has low production cost, has low requirement on production equipment, and has simple and flexible immunization mode, and is conducive to large-scale popularization and use in the market.

Description

Technical Field

[0001] This invention belongs to the field of veterinary vaccine strain technology, and in particular relates to a Newcastle disease virus vector vaccine strain expressing C-type aMPV F protein and G protein and its application. Background Technology

[0002] Avian metapneumovirus (aMPV) is an acute, highly contagious upper respiratory tract infection in poultry caused by avian metapneumovirus (aMPV), also known as Turkey rhinotracheitis virus (TRTV) or avian pneumovirus (APV). Primarily transmitted through the air, aMPV is the most impactful respiratory disease on turkeys after avian influenza, causing severe economic losses to poultry farming worldwide. The disease was first reported in South Africa in 1980, followed by reports in France and the United Kingdom. Since then, aMPV has been reported in most countries and regions worldwide. my country first isolated and reported the virus in 1998. In recent years, aMPV infection has been reported in various regions of my country, and it is widespread among poultry. aMPV is mainly transmitted through the air, infecting chickens, turkeys, ducks, and wild birds, and is associated with Swollen head syndrome (SHS) in chickens. This disease primarily causes acute upper respiratory tract infections and can also affect egg quality, leading to a rapid decline in egg production. Avian metapneumovirus (aMPV) infection in poultry is often accompanied by secondary bacterial infections, which can increase mortality by 25%-40%, causing severe economic losses to the poultry industry. Based on the nucleotide sequence of the G protein encoded by aMPV, it can be divided into four subtypes: A, B, C, and D. Systematic sequence analysis revealed that aMPV types A, B, and D are more homologous than aMPV type C, which is genomically similar to human metapneumovirus. In 2013, aMPV-C infection was first reported in chickens in my country; in 2014, aMPV-C was first isolated from Muscovy ducks in my country, and it was confirmed that aMPV-C can cause a decrease in egg production in Muscovy ducks, resulting in respiratory and ovarian-oviduct symptoms.

[0003] Vaccination and strict biosafety measures are the main measures for controlling aMPV disease. Inactivated and live attenuated aMPV vaccines for turkeys and chickens are commercially available. Studies have shown good cross-protection between aMPV-A and aMPV-B vaccines, and they also provide partial protection against subtype C aMPV. However, the aMPV-C vaccine does not provide protection against subtypes A and B. Cell-cultured live attenuated vaccines for aMPV-A and aMPV-B subtypes are widely used worldwide, but the aMPV-C subtype vaccine is only used in the United States. Inactivated aMPV vaccines can induce highly effective and durable antibodies, but they need to be used in combination with live attenuated vaccines; using inactivated vaccines alone is not ideal and cannot provide complete protection. Live attenuated vaccines can stimulate systemic immunity and local respiratory immunity. Upon initial immunization with aMPV live vaccines, the humoral immune response in turkeys is very weak, a phenomenon more pronounced in chickens, but cellular immunity generated through the respiratory tract still provides protection. Although live attenuated vaccines have relatively good protective effects, there are still many problems: the duration of immunity is short, requiring repeated vaccinations, which can lead to the spread of attenuated strains and frequent infections in vaccinated areas; live attenuated aMPV vaccines have some immunosuppression, which may cause secondary bacterial infections and other viral infections; long-term use of live attenuated aMPV vaccines may cause virulence reversion.

[0004] Newcastle disease virus (NDV), a member of the Paramyxoviridae family, has become a favored viral vaccine vector. Numerous studies have shown that recombinant NDV vector vaccines induce faster and better humoral and mucosal immune responses than conventional vaccines. In recent years, recombinant NDV has successfully expressed exogenous proteins including: chicken infectious bursal virus VP2 protein, H5 subtype avian influenza virus HA protein, human immunodeficiency virus Gag protein, chicken infectious laryngotracheitis gB / gD protein, rabies G glycoprotein, and novel coronavirus S protein. NDV offers significant advantages as a vector vaccine: NDV is an RNA virus derived from birds, and due to its strictly limited host range, it is highly attenuated in humans and other animals, making it safe for use in mammals; its viral genome is relatively small, allowing for artificial modification, and the technology is relatively mature; NDV replication occurs only in the cytoplasm, avoiding contact with the host genome, reducing the risk of recombination and ensuring good vector stability; the virus can grow at high titers in chicken embryos and many cell lines, reducing production costs; the small NDV genome encodes only a few proteins, facilitating the expression and display of exogenous proteins; NDV can be naturally transmitted through the respiratory tract, thus inducing not only humoral and cellular immunity but also mucosal immunity; Newcastle disease virus can be administered via drinking water or spray, saving considerable manpower and resources, which is beneficial for the widespread use of vector vaccines. However, the relatively small size of the Newcastle disease virus genome means that excessively long inserted exogenous genes can affect viral replication, as well as the stability and expression of the exogenous genes, limiting the widespread use of Newcastle disease virus vector vaccines. Summary of the Invention

[0005] This invention provides a Newcastle disease virus vector vaccine strain expressing type C aMPV F and G proteins and its application. This vector vaccine has good immunization efficacy, inducing humoral immunity, cellular immunity, and mucosal immunity, providing comprehensive protection for immunized animals. It can provide complete protection against both aMPV-C wild-type strain and NDV-VII wild-type strain. The production cost is low, the requirements for production equipment are not high, and the immunization method is simple and flexible, which is conducive to large-scale market promotion and use.

[0006] To achieve the above objectives, the present invention provides a Newcastle disease virus vector vaccine strain expressing type C aMPV F and G proteins. An infectious clone plasmid of NDV is constructed using a type VII Newcastle disease virus rNDV strain as the host and genetically rescued. The infectious clone plasmid has nucleotide sequences of F gene, G gene, or tandem F gene and G gene inserted at different positions of NDV.

[0007] The Newcastle disease virus rNDV strain of genotype VII was deposited at the China Center for Type Culture Collection on September 27, 2023, with accession number CCTCC NO: V202391.

[0008] Preferably, nucleotide sequences of the F and G genes are inserted in tandem between the P and M genes of NDV, or the F gene is inserted between the P and M genes of NDV and the G gene is inserted between the M / F or F / HN genes.

[0009] During the implementation of this invention, it was discovered that inserting the F gene between the P / M genes and the G gene between the M / F genes of NDV resulted in extremely low growth titers in the rescued virus, making it unsuitable as a vaccine candidate strain. Therefore, this invention selects to insert the F gene between the P / M genes and the G gene between the F / HN genes to overcome the deficiencies or shortcomings of the prior art. When using the original, unmodified G gene as the exogenous protein expression gene, the G protein is almost not expressed or the expression level is too low to reach a detectable level. To overcome the above defects, this invention replaces the nucleotide sequences encoding the N-terminal intracellular and transmembrane regions of the G protein with the nucleotide sequences encoding the N-terminal intracellular and transmembrane regions of the HN protein in the NDV vector. Specifically, the 54 amino acids at the N-terminus of the G protein (G gene 1-162nt) are replaced with the 45 amino acids at the N-terminus of the NDV HN protein (HN gene 1-135nt). The expression level of the G protein is significantly improved, achieving the expected effect and requirements of the vector vaccine.

[0010] It is understood that the G genes mentioned in this invention are all modified genes as described above, and the G proteins in the vector vaccines are all proteins expressed by modified G genes.

[0011] As a preferred embodiment, the nucleotide sequence of the F gene is shown in SEQ ID NO: 1, the nucleotide sequence of the G gene is shown in SEQ ID NO: 2, and the full genome sequence of the Newcastle disease virus rNDV strain of genotype VII is shown in SEQ ID NO: 3.

[0012] Preferably, the F gene and the G gene are linked together by the IRES gene, the sequence of which is shown in SEQ ID NO: 4.

[0013] Preferably, the Newcastle disease virus vector vaccine strain is rNDV-FG or rNDV-F / IRES / G, both of which provide complete protection against both aMPV-C and genotype VII Newcastle disease virus.

[0014] Preferably, the Newcastle disease virus vector vaccine strain is a live attenuated vaccine.

[0015] The present invention also provides the application of the Newcastle disease virus vector vaccine strain according to any one of the technical solutions in the prevention or treatment of highly contagious avian hemiplegic disease caused by avian hemiplegic virus.

[0016] Preferably, the poultry is selected from domestic poultry such as chickens, turkeys, and ducks, as well as wild birds, with Muscovy ducks, semi-Muscovy ducks, or Cherry Valley ducks being the most preferred.

[0017] As a preferred method, the inoculation method is used;

[0018] The vaccine strain rapidly induces an immune dose of 10 to produce neutralizing antibodies. 7.0 TCID 50 On day 28 after the first immunization with rNDV-FG strain or rNDV-F / IRES / G(P / M) strain, the neutralizing antibody levels were 2. 8.6 and 2 8.0 .

[0019] The present invention also provides a commercially available aMPV vaccine, prepared using the Newcastle disease virus vector vaccine strain described in any of the above technical solutions.

[0020] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0021] 1. The Newcastle disease virus vector vaccine strain provided by this invention can fill the gap in the domestic market for aMPV vaccines.

[0022] 2. The Newcastle disease virus vector vaccine strain provided by this invention can solve the safety issues of aMPV vaccines. aMPV inactivated or subunit vaccines require adjuvants and necessitate large doses or multiple immunizations to achieve the desired immune effect, easily causing stress or adverse reactions in immunized animals. aMPV attenuated live vaccines are prone to virulence reversion when used in the field and may also cause immunosuppression, affecting the immunization efficacy of other vaccines and posing potential safety risks. The Newcastle disease virus vector, genetically modified, has been verified to be a low-virulence strain with a relatively stable genome, preventing virulence reversion. Furthermore, immunization can be administered via spraying, reducing stress caused by handling immunized animals.

[0023] 3. The Newcastle disease virus vector vaccine strain provided by this invention can solve the efficacy problem of aMPV vaccines. Inactivated or subunit aMPV vaccines have limited immunogenicity and cannot provide complete protection against virulent strains when used alone. The Newcastle disease vector vaccine combines adjuvant and vaccine effects, stimulating the body to produce humoral, cellular, and mucosal immunity, providing comprehensive protection against virulent aMPV strains. Moreover, its immunogenicity is long-lasting and provides good protection against different heterologous strains. Furthermore, compared to conventional attenuated live NDV vaccines such as the LaSota strain, Clone 30 strain, and VGGA strain, genotype VII NDV has a stronger ability to invade cells and tissues, and can activate a stronger systemic immune response while ensuring safety.

[0024] 4. This invention, based on a Newcastle disease virus vector vaccine strain, addresses the issue of the impact of inserted exogenous genes on Newcastle disease virus proliferation and exogenous gene expression. Through continuous optimization, the impact of exogenous genes on Newcastle disease virus proliferation is minimized, and the stability of exogenous gene expression is improved.

[0025] 5. The present invention develops a dual vaccine strain based on the Newcastle disease virus vector vaccine strain, which can simultaneously provide complete protection against both aMPV-C and NDV-VII wild-type strains. Attached Figure Description

[0026] Figure 1 The PCR identification results of the rNDV-aMPV recombinant virus provided in the embodiments of the present invention are as follows: 1. 200bp DNA Ladder Marker; 2. Amplification of the F gene of rNDV-F strain; 3. Amplification of the G gene of rNDV-G strain; 4. Amplification of the F-IRES-G gene of rNDV-F / IRES / G(P / M) strain; 5. Amplification of the F gene of rNDV-FG strain; 6. Amplification of the G gene of rNDV-FG strain.

[0027] Figure 2 Comparison of the intraembryo growth kinetics of recombinant viruses provided in embodiments of the present invention;

[0028] Figure 3 The immunofluorescence assay provided in this embodiment of the invention is used to identify the expression of exogenous genes.

[0029] Figure 4 The results of neutralizing antibody detection in Muscovy duck serum provided in this embodiment of the invention;

[0030] Figure 5 The result of Muscovy duck NDV antibody detection (HI) provided in the embodiment of the present invention. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0032] Example 1: Construction of a recombinant Newcastle disease virus vector vaccine expressing the F and G genes of Muscovy duck type C avian metapneumovirus

[0033] Using gene recombination technology, the F gene of aMPV, the modified G gene (hereinafter referred to as G gene), and the IRES sequence were cloned into the vector pBluescript II KS(+) to construct the plasmid pBlu-F-IRES-G. The F gene (its nucleotide sequence is shown in SEQ ID NO: 1), the G gene (its nucleotide sequence is shown in SEQ ID NO: 2), or the F-IRES-G gene were inserted between the P / M genes or between the F / HN genes of the genotype VII Newcastle disease virus rNDV strain (its full genome sequence is shown in SEQ ID NO: 3) to construct an infectious cloning plasmid of NDV and rescue the recombinant virus.

[0034] Primers were designed based on the F and G gene sequences of avian metapneumovirus type C isolated and preserved in the laboratory, the whole genome sequence of Newcastle disease virus type VII rNDV strain, and the synthesized IRES gene sequence (as shown in SEQ ID NO: 4), and five infectious cloning plasmids were constructed. Primer and gene synthesis were completed by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are shown in Table 1.

[0035] Table 1 Primer sequences used for constructing infectious clones of Newcastle disease virus vector vaccines

[0036]

[0037]

[0038] The specific steps are described below.

[0039] 1.1 Construction of the MPV F-IRES-G tandem expression cassette

[0040] Using aMPV cDNA and the modified G gene as templates, the F and G genes were amplified using primers NDV-F / G-LF / NDV-F / G-LR and NDV-F / G-RF / NDV-F / G-RR, respectively. The synthesized IRES sequence was amplified using primers NDV-F / G-MF / NDV-F / G-MR, also as a template. The vector plasmid pBluescript II KS(+) was double-digested with EcoRI / XhoI and then combined with the three PCR-amplified fragments for gel electrophoresis and recovery. The recovered four gene fragment products were mixed in a 1:1:1:1 ratio (2.5 μL each) according to... Instructions for using high-fidelity DNA assembly premix: Add 10 μL HiFi DNA Assembly Master Mix was mixed thoroughly and incubated at 50°C for 1 hour before being transformed into E. coli competent cells JM109. Positive clones were sequenced correctly and stored. The constructed plasmid was named pBlue-F / IRES / G.

[0041] 1.2 Construction of infectious clones of Newcastle disease virus containing the aMPV F or G gene

[0042] Using aMPV cDNA as a template, the F gene was amplified using primers NDV-FF / NDV-FR; plasmid PBRT-NDV-pme I (P / M) was digested with pme I. Positive bands were recovered after gel electrophoresis, and... The instructions for the high-fidelity DNA assembly premix were followed to assemble the fragments and construct the plasmid PBRT-NDV-F.

[0043] Using the modified G gene as a template, the G gene was amplified with primers NDV-GF / NDV-GR; plasmid PBRT-NDV-pmeI (P / M) was digested with pmeI. Positive bands were recovered after gel electrophoresis, and... The instructions for the high-fidelity DNA assembly premix were followed to assemble the fragments and construct the plasmid PBRT-NDV-G.

[0044] Using plasmid pBlue-F / IRES / G as a template, the F / G tandem gene was amplified using primers NDV-F / G-AF / NDV-F / G-AR; ​​plasmid PBRT-NDV-pme I (P / M) was digested with pme I. Positive bands were recovered after gel electrophoresis, and... Follow the instructions for the high-fidelity DNA assembly premix to assemble fragments and construct the plasmid PBRT-NDV-F / G(P / M).

[0045] Using plasmid pBlue-F / IRES / G as a template, the F / G tandem gene was amplified using primers NDV-F / G-AF-2 / NDV-F / G-AR-2; plasmid PBRT-NDV-pme I (F / HN) was digested with pme I. Positive bands were recovered after gel electrophoresis, and the gene was analyzed according to... Following the instructions for the high-fidelity DNA assembly premix, fragments were assembled to construct the plasmid PBRT-NDV-F / G(F / HN).

[0046] Using aMPV cDNA as a template, the F gene was amplified with primers NDV-FF-2 / NDV-FR-2; plasmid PBRT-NDV-pacI / pme I was digested with pacI I. Positive bands were recovered after gel electrophoresis, and... Following the instructions for the high-fidelity DNA assembly premix, fragments were assembled to construct plasmid PBRT-NDV-F-pme I. Using the modified G gene as a template, the G gene was amplified with primers NDV-GF-2 / NDV-GR-2. Plasmid PBRT-NDV-F-pme I was digested with pme I enzyme. The G gene was cloned into the F / HN gene region using the same method to finally obtain plasmid PBRT-FG.

[0047] 1.3 Transfection to obtain recombinant Newcastle disease virus

[0048] BSR cells were seeded in 6-well cell culture plates and transfected when the cells reached 80% confluency. 5 μg of infectious clonal plasmids PBRT-NDV-F, PBRT-NDV-G, PBRT-NDV-F / G (P / M), PBRT-NDV-F / G (F / HN), and PBRT-FG were mixed with three helper plasmids (PCI-NP 2.5 μg, PCI-P 1.25 μg, PCI-L 1.25 μg) and co-transfected according to the calcium phosphate transfection kit instructions. 48 h post-transfection, the transfected cells were frozen and thawed, and the supernatant was collected and inoculated into 9-day-old SPF chicken embryos. After 120 h of culture, the allantoic fluid from the chicken embryos was collected, and the HA titer was determined. HA-positive samples were cryopreserved; HA-negative samples were further blindly passaged for 3 generations. The rescued recombinant NDV strains expressing the aMPV antigen gene were named rNDV-F, rNDV-G, rNDV-F / IRES / G(P / M), rNDV-F / IRES / G(F / HN), and rNDV-FG, respectively. Their PCR identification results are as follows: Figure 1 As shown.

[0049] The allantoic fluid of chicken embryos collected 120 hours after embryo transfer had an HA titer of 2. 4 -2 8 After dilution of the allantoic fluid, chicken embryos were inoculated again, and the HA titer stabilized at 2. 8 -211 The virus was passaged for 15 generations, with viral allantoic fluid samples collected every 5 generations. RNA was extracted and reverse transcribed into cDNA. The inserted genes of the five recombinant viruses were sequenced. Primers P / M-J1 / P / M-J2 amplified the inserted sequence between the P and M genes, and primers F / HN-J1 / F / HN-J2 amplified the inserted sequence between the F and HN genes. The results showed that the inserted aMPV genes had no deletions or mutations. This indicates that the rescued NDV vector vaccine strains can be stably inherited.

[0050] Example 2: Determination of mean lethal time (MDT), intracerebral pathogenicity index (ICPI), and intravenous pathogenicity index (IVPI) in chicken embryos containing recombinant Newcastle disease virus.

[0051] The values ​​of MDT, ICPI, and IVPI can directly reflect the pathogenicity of Newcastle disease virus. Therefore, the above three indices were measured on the five recombinant Newcastle disease virus vector vaccine strains and the parental rNDV strain, and the virulence of NDV was determined according to the OIE standard. The results are shown in Table 2.

[0052] Table 2. Determination of pathogenicity index of NDV vector vaccine strains

[0053]

[0054] Note: rNDV is the parental strain without the insertion of the foreign gene.

[0055] The results showed that all five recombinant NDV vector vaccine strains were low-virulence strains, meeting the standards for use as attenuated live vaccines.

[0056] Example 3: Evaluation of the growth kinetics of the recombinant NDV vector vaccine strain in chicken embryos

[0057] The allantoic fluids from the collected parental rNDV strain and 5 recombinant vector vaccine strains were mixed at a ratio of 10:1. 5.0 EID 50 0.1 ml of allantoic fluid was inoculated into the allantoic cavity of SPF chicken embryos. Allantoic fluid was harvested at 24 h, 48 h, 72 h, and 96 h post-inoculation. At each time point, six SPF chicken embryos were randomly collected, and the EID of six different viruses per milliliter of allantoic fluid was determined. 50 Plot the growth curve, such as Figure 2 As shown.

[0058] like Figure 2As shown, the growth characteristics of rNDV-F, rNDV-G, and rNDV-FG strains are similar to those of the parental rNDV strain, indicating that the insertion of the aMPV antigen gene did not affect the growth rate of NDV, which still maintained high-titer growth characteristics. However, the proliferation rates of rNDV-F / IRES / G (P / M) and rNDV-F / IRES / G (F / HN) strains decreased slightly, indicating that the insertion of the F and G genes into the NDV genome had a slight impact on the NDV proliferation rate. It is speculated that since both aMPV and NDV are avian viruses, their genes have good compatibility, and the insertion of the aMPV gene into the Newcastle disease virus genome has a relatively small impact on Newcastle disease virus replication.

[0059] Example 4: Indirect immunofluorescence assay to identify the expression of the aMPV exogenous gene.

[0060] When CEF cells in a 96-well plate reached 90% confluence, the culture medium was discarded, and 100 μL of recombinant virus (1:100 dilution) was added to each well. After incubation at 37°C for 2 hours, 100 μL of DMEM medium containing 4% FBS was added, and the cells were cultured for 48 hours before indirect immunofluorescence identification. The culture medium was discarded, and the cells were washed three times with PBS. 100 μL of cold anhydrous ethanol was added to each well for fixation, and the cells were fixed at 4°C for 2 hours. The fixative was discarded, and the cells were washed three times with PBST. 100 μL of primary antibody (NDV polyclonal antibody serum, aMPV-immunized duck polyclonal antibody serum, 1:100 dilution) was added to each well. After incubation at room temperature for 1 hour, the cells were washed three times with PBST. 100 μL of FITC-labeled secondary antibody (rabbit anti-chicken, goat anti-duck, 1:500 dilution) was added to each well. The cells were incubated at room temperature in the dark for 30 minutes, the liquid was discarded, and the cells were washed three times with PBST. Protein expression was observed under a fluorescence microscope. Figure 3 As shown.

[0061] Depend on Figure 3As shown, the negative control showed no visible fluorescence; in the rNDV group, only the wells incubated with NDV antibodies showed green fluorescence, while the wells incubated with aMPV antibodies showed no fluorescence; in the five recombinant virus groups, both the NDV antibody and aMPV antibody wells showed green fluorescence. Among all groups except the negative control, the NDV antibody wells showed the brightest fluorescence; in the aMPV antibody wells, the single-gene expression groups rNDV-F and rNDV-G showed similar fluorescence virulence to the double-gene expression groups rNDV-F / IRES / G(P / M), rNDV-F / IRES / G(F / HN), and rNDV-FG, indicating that the exogenous gene can be expressed regardless of whether a single F gene or G gene is inserted into the NDV genome, or whether two genes are inserted simultaneously. Considering that the range of antibodies induced by the expression of exogenous proteins through double gene insertion is broader and the protective effect may be better, three recombinant viruses, rNDV-F / IRES / G(P / M), rNDV-F / IRES / G(F / HN), and rNDV-FG, were selected for further animal experiments.

[0062] Example 5: Evaluation of the Immunization Efficacy of Recombinant NDV Vector Vaccine in Muscovy Ducklings

[0063] One-day-old Muscovy ducklings were randomly divided into 6 groups of 10 each. Before immunization, antigen and antibody levels for NDV and aMPV were measured, and all results were negative. At 7 days of age, the ducklings were inoculated with 0.1 ml of recombinant virus rNDV-F / IRES / G(P / M), rNDV-F / IRES / G(F / HN), rNDV-FG, and the parental rNDV strain via ocular injection. In the other two groups, one group was immunized intramuscularly with one dose (0.5 ml) of aMPV-C inactivated vaccine (prepared by the R&D center laboratory of Qingdao Yibang Biotechnology Co., Ltd., with a virus content of 10 mg / dose before inactivation). 5.0 TCID 50 One group was inoculated with the same dose of PBS via eye drops as a negative control. All ducks were isolated and given a booster immunization at the same method and dose 14 days after the first immunization. Blood samples were collected from the jugular vein at 7, 14, 21, and 28 days after the first immunization. Serum was separated, and the levels of neutralizing antibodies against aMPV and HI antibodies against NDV were measured. Twenty-eight days after the first immunization, ducks were challenged with either wild-type aMPV-C or wild-type NDV-VII strain via eye drops. They were observed for 14 days after challenge. Laryngeal and cloacal swabs were collected on days 3, 5, and 7 after challenge. The shedding of aMPV-C and NDV-VII was measured using quantitative real-time PCR, and the results were statistically analyzed.

[0064] The method for determining aMPV-C neutralizing antibodies is as follows: Collected serum was inactivated in a 56℃ metal bath for 30 min before detection. 50 μL of serum-free DMEM was added to each well of a 96-well cell culture plate. Serum samples were serially diluted 2-fold until a final dilution of 1:2048 was achieved, with each dilution repeated in quadruplicate. Except for the negative control wells, 50 μL of aMPV-C virus (containing 200 TCID50) was added to each well. 50 The virus was incubated at 37°C for 1 hour. 100 μL of Vero cells (6 x 10⁻⁶ cells) were added to each well. 4.0 Cells were incubated at 37°C for 5-7 days. The cell status of each well was observed daily, and the number of diseased cells in each well was recorded. The neutralizing antibody titer was calculated using the Spearman-Karber method, and the average value was used to plot a neutralizing antibody curve. The NDV HI antibody level was determined according to standard methods.

[0065] The specific grouping and immunization details for the animal experiments are shown in Table 3.

[0066] Table 3. Grouping of Muscovy ducklings after immunization and challenge

[0067]

[0068] Neutralizing antibody test results showed that all three groups immunized with the recombinant NDV vector vaccine could induce the production of neutralizing antibodies against aMPV-C. Figure 4 Furthermore, the level of neutralizing antibodies was highest 28 days after the first immunization, with the rNDV-FG group exhibiting the highest induced neutralizing antibody levels, averaging 2. 8.6 The second group was the rNDV-F / IRES / G(P / M) group, with an average neutralizing antibody level of 2. 8.0 The rNDV-F / IRES / G(F / HN) immunization group induced the lowest level of neutralizing antibodies, with an average neutralizing antibody level of 2. 7.4 Furthermore, immunization of Muscovy ducklings with rNDV-FG, rNDV-F / IRES / G (P / M), and rNDV-F / IRES / G (F / HN) rapidly induced the production of neutralizing antibodies, with low levels detectable as early as 7 days post-immunization. No neutralizing antibodies were produced in the rNDV and PBS immunization groups. Neutralizing antibodies were also produced in the aMPV-C inactivated vaccine immunization group, but at a slower rate, with low levels detectable only 14 days after the initial immunization. At 28 days, the neutralizing antibody levels were significantly lower than those induced by the three recombinant Newcastle disease virus vector vaccines described in this invention. No neutralizing antibodies against aMPV-C were produced in the PBS and rNDV immunization groups.

[0069] NDV HI assay results showed that rNDV-F / IRES / G (P / M), rNDV-F / IRES / G (F / HN), rNDV-FG, and the rNDV immunization group could all induce HI antibodies against NDV. Titer was detectable 7 days after the first immunization, with the highest HI titer at 28 days. Antibody levels showed a continuous upward trend, and there were no significant differences between the groups. No HI antibodies were produced in the PBS immunization group and the aMPV-C inactivated vaccine immunization group. Figure 5 ).

[0070] The results of the challenge experiments showed that during the 14-day observation period after challenge, no Muscovy ducks died, regardless of whether they were challenged with aMPV-C or NDV-VII wild-type strains, which may be related to the age of the ducks. However, some Muscovy ducks did become ill. In the aMPV challenge group, the sick ducks exhibited symptoms such as coughing, runny nose, and tracheal rales, generally developing symptoms 5 days after challenge and gradually recovering around 10 days later. Swab test results at 3, 5, and 7 days post-challenge showed that in the three recombinant NDV vector vaccine immunization groups, no viral shedding was detected in the rNDV-FG and rNDV-F / IRES / G(P / M) immunization groups. However, in the rNDV-F / IRES / G(F / HN) immunization group, viral shedding was detected in one duck's throat swab on day 3 post-challenge and in two ducks' throat swabs on day 5. In the aMPV-C inactivated vaccine immunization group, viral shedding was detected in two ducks' throat swabs on day 3 post-challenge, and in throat swabs from three ducks and cloacal swabs from two ducks on day 5. Viral shedding was detected in both the rNDV and PBS immunization groups at 3, 5, and 7 days post-challenge.

[0071] In the NDV-VII challenge group, sick ducks exhibited lethargy, yellowish-white, white, or yellowish-green watery feces, and nasal discharge or tearing. Symptoms generally appeared 3 days after challenge and gradually improved after about 7 days. Swab tests at 3, 5, and 7 days post-challenge showed no viral shedding in the three recombinant NDV vector vaccines and the rNDV immunization group, indicating that these four viruses provided complete protection against NDV-VII challenge, and the insertion of foreign genes into the NDV genome did not affect the NDV immunization effect. However, viral shedding was observed in both the aMPV-C inactivated vaccine immunization group and the PBS immunization group.

[0072] Table 4. Disease incidence and virus shedding in experimental ducks after challenge.

[0073]

[0074]

[0075] In summary, the rNDV-FG and rNDV-F / IRES / G(P / M) vector vaccine strains exhibited good proliferation efficiency on chicken embryos, induced high levels of neutralizing antibodies after immunization, and provided the best immune protection after challenge. They can simultaneously provide complete protection against both aMPV-C and NDV-VII, making them suitable as vaccine candidate strains.

Claims

1. A Newcastle disease virus vector vaccine strain expressing type C aMPV F and G proteins, characterized in that, An infectious clone plasmid of NDV was constructed using Newcastle disease virus rNDV strain genotype VII as the host and genetically rescued. The nucleotide sequences of F gene, G gene, or tandem F gene and G gene were inserted at different positions of NDV in the infectious clone plasmid. The Newcastle disease virus rNDV strain of genotype VII was deposited at the China Center for Type Culture Collection on September 27, 2023, with accession number CCTCC NO: V202391. Insert nucleotide sequences of the F and G genes in tandem between the P / M genes of NDV, or insert the F gene between the P / M genes of NDV and the G gene between the F / HN genes. The nucleotide sequence of the G gene is shown in SEQ ID NO:

2.

2. The Newcastle disease virus vector vaccine strain according to claim 1, characterized in that, The nucleotide sequence of the F gene is shown in SEQ ID NO: 1; The complete genome sequence of Newcastle disease virus rNDV strain genotype VII is shown in SEQ ID NO:

3.

3. The Newcastle disease virus vector vaccine strain according to claim 1, characterized in that, The F gene and G gene are linked together via the IRES gene, the sequence of which is shown in SEQ ID NO:

4.

4. The use of the Newcastle disease virus vector vaccine strain according to any one of claims 1-3 in the preparation of a drug for the prevention of highly contagious avian hemiplegic disease caused by avian hemiplegic virus, characterized in that, The drug is for Muscovy ducks, semi-Muscovy ducks, or Cherry Valley ducks.

5. A commercially available aMPV vaccine, characterized in that, The vaccine was prepared using the Newcastle disease virus vector vaccine strain described in any one of claims 1-3.

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