A novel attenuated strain of goose parvovirus and its construction method and application

Abstract

The present invention provides a novel attenuated strain of goose parvovirus and its construction method and application, belonging to the field of virus vaccines. The present invention has determined two artificial attenuation sites that weaken the virulence of the novel goose parvovirus through research. The NGPV-NMG50 attenuated strain obtained in this application is used as a live vaccine prepared as an immunogen, and the feather loss protection rate of ducks can reach 100% without the addition of adjuvants. The minimum lethal dose of the virus is determined by diluting the NMG21 wild strain in multiples, and then the optimal immune dose of the NGPV-NMG50 attenuated strain is screened, and the optimal immune dose results of the NGPV-NMG50 attenuated strain in clinical use are obtained, so that ducklings can effectively establish an immune barrier against the novel goose parvovirus.

Description

Technical Field

[0001] The invention belongs to the field of virus vaccines, and in particular relates to a novel attenuated strain of goose parvovirus and a construction method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Waterfowl Parvovirus is a type of non-enveloped single-stranded DNA virus, with a host range that includes geese, Muscovy ducks and ducks. The new goose parvovirus (NGPV) is a new type of waterfowl parvovirus that has been mutated from the traditional goose parvovirus since 2015 and specifically infects ducks. The pathogen spreads rapidly in China, with a wide range of epidemics and no specific biological products for prevention and control. Once the ducks are infected, they cannot be treated with drugs. The current large-scale epidemic of the disease has caused huge economic losses to my country's duck farming industry.

[0004] The traditional goose parvovirus is also known as gosling plague, and its main pathological feature is the appearance of fibrinous intestinal plugs in the small intestine of goslings. Although the new goose parvovirus originated from the goose parvovirus, the two have significant differences in their pathogenicity to their respective hosts. After the ducks are infected with the new goose parvovirus, the clinical manifestations are developmental delay, beak atrophy, and tongue protrusion, commonly known as macroglossia or "short-beaked dwarf syndrome". The disease mainly occurs in ducklings under 3 weeks of age, with a low mortality rate, but the elimination rate of disabled ducks is as high as 80%. The diseased ducklings are short, with a significantly increased feed-to-meat ratio, and they often have tibia and wing bone fractures when they grow up. The average weight of the diseased ducks is 20% - 30% lower than that of the normal market, and in severe cases it is only 50% of the normal weight.

[0005] Since 2021, new infection characteristics have appeared in ducks infected with the new goose parvovirus. In addition to obvious short-beak dwarfism syndrome, the infected ducks also showed new infection characteristics including feather loss on the back, thinning of the small intestinal wall, and intestinal embolism at the junction of the cecum and ileum (the site of yolk absorption). The virus caused a mortality rate of 40% in the ducks. This result shows that the new goose parvovirus has undergone a large-scale mutation during the epidemic, and this mutation may also affect the effectiveness of some biological products developed for the virus in the past.

[0006] Attenuated vaccine vaccination is an effective means of preventing and controlling waterfowl parvovirus. Currently, the continuous mutation of the new goose parvovirus has led to a lack of effective live vaccine products against the pathogen on the market, greatly increasing the risk of infection by the virus in the duck farming industry.

[0007] In addition, the development of live vaccine products requires subculture of wild strains to obtain weak strains with reduced virulence. When culturing strong strains in large quantities, the production environment needs to be strictly controlled to prevent the spread of strong strains. Therefore, it is of great significance to artificially mutate the virulence sites in the wild strains of new goose parvovirus through genetic engineering to reduce the virulence of the virus and develop a new goose parvovirus live vaccine prepared with weak strains. Summary of the invention

[0008] In view of the above-mentioned prior art, the purpose of the present invention is to provide a novel attenuated strain of goose parvovirus and a construction method and application thereof.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a novel attenuated strain of goose parvovirus, characterized in that the attenuated strain is obtained by subjecting the VP1 protein of the novel wild-type goose parvovirus to site-directed mutagenesis at amino acids 78 and 567.

[0011] The amino acid sequence of the attenuated strain VP1 protein is shown in SEQ ID NO:4.

[0012] The novel goose parvovirus wild strain is NGPV-NMG21 strain, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​October 24, 2024, with the deposit name: NGPV-NMG21, and the classification name: novel goose parvovirus ( Anser anser novus parvovirus ), the deposit number is: CGMCC No.46205.

[0013] The second aspect of the present invention provides a method for constructing the attenuated strain, which is characterized by comprising the following steps:

[0014] Through genetic engineering, site-directed mutations were performed on the 233rd and 1700th nucleotides of the VP1 gene of the new wild-type goose parvovirus strain to obtain a weak strain with reduced virulence.

[0015] Preferably, the site-directed mutagenesis is to mutate the nucleotide at position 233 of the VP1 genome of the novel wild-type goose parvovirus from G to A and the nucleotide at position 1700 from G to C.

[0016] The VP1 genome nucleotide sequence of the attenuated strain obtained after the site-directed mutagenesis is shown in SEQ ID NO:2.

[0017] The third aspect of the present invention provides the use of the attenuated strain in the preparation of a vaccine for preventing or treating a novel goose parvovirus.

[0018] The vaccine is a live attenuated vaccine.

[0019] The novel goose parvovirus vaccine is prepared by the following steps:

[0020] The weak strain of the novel goose parvovirus is inoculated into duck embryo fibroblast DEF for culture, and the culture fluid containing the immune effective amount is used as an immunogen to prepare a vaccine for preventing or treating the novel goose parvovirus.

[0021] The term "immunologically effective amount" refers to the amount of avirulent novel goose parvovirus strain that will induce an immune response against the novel goose parvovirus."Immunologically effective amount" will depend on factors such as the species, breed, age, and health status of the recipient animal.

[0022] In one embodiment of the present invention, the minimum lethal dose of the virus is determined by diluting the wild strain NMG21 in multiples, and the optimal immune dose of the weak strain NGPV-NMG50 is screened using the minimum lethal dose of the wild strain to obtain the optimal immune dose of the weak strain NGPV-NMG50 for clinical use.

[0023] The vaccine also includes a pharmaceutically acceptable adjuvant.

[0024] The beneficial effects of the present invention include:

[0025] 1. The present invention provides two artificial attenuation sites for weakening the virulence of a novel wild strain of goose parvovirus, namely, a nucleotide mutation at position 233 of the VP1 genome of the wild strain, which will cause the amino acid encoded by the site to change from AGC (serine) to AAC (asparagine); and a nucleotide mutation at position 1700 of the VP1 genome of the wild strain, which will cause the amino acid encoded by the site to change from GGA (glycine) to GCA (alanine), laying a solid foundation for the development of a novel live goose parvovirus vaccine prepared with a weakened strain.

[0026] 2. The artificial attenuation sites provided in this application can be used to carry out genetic engineering modification of wild strains, and the strains with weakened virulence can be cultured and subcultured to prepare vaccines, thereby avoiding the risk of strong toxic spread caused by the use of strong strains for subculture, improving biosafety and reducing production costs.

[0027] 3. The novel goose parvovirus NGPV-NMG50 weak strain obtained by the present invention provides a valuable biological sample for studying the virulence gene loci of waterfowl parvovirus. Compared with the wild strain, the strain titer is increased from 10 -7.3 / 0.2 mL down to 10 -4.7 / 0.2 mL, the NGPV-NMG50 weak strain has no virulence reversion phenomenon, and has good stability. The live vaccine with the weak strain NGPV-NMG50 as the immunogen has good safety. The ducks injected with the vaccine did not show any local and systemic adverse reactions caused by the vaccine. No adjuvant is needed to produce 100% immune protection for ducks.

[0028] 4. The present invention screened the optimal immune dose of the weak strain of NGPV-NMG50 for clinical use, enabling ducklings to effectively establish an immune barrier against the new goose parvovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein:

[0030] Figure 1 The figure shows the viral growth curve of NGPV-NMG21 and NGV-NMG50 measured using the qPCR method.

[0031] Figure 2 This is an indirect immunofluorescence detection image. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0033] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present application, rather than to limit the scope of the present invention.

[0034] As mentioned earlier, the large-scale epidemic of the new goose parvovirus has caused huge economic losses to my country's duck farming industry. Since 2021, new infection characteristics have appeared in duck flocks infected with the new goose parvovirus. Such mutations may also have affected the effectiveness of some biological products developed for the virus in the past. At present, the continuous mutation of the new goose parvovirus has led to a lack of effective vaccine products against the pathogen on the market. The development of new goose parvovirus vaccines or other immune products is of great significance for disease prevention and control.

[0035] Based on this, the present invention provides a novel weak strain of goose parvovirus and its construction method and application. Through analysis, the novel goose parvovirus exists in an icosahedral symmetrical structure formed by a capsid with a diameter of about 20 to 22 nm, and usually uses the hairpin structure at the end of its own genome to complete the DNA replication of the autonomous virus. The full length of the viral genome consists of about 5058 nucleotides, in which the stem-loop structures at both ends are responsible for ensuring the initiation and efficiency of viral replication. The virus is an inclusive protein structure, in which the VP1 protein contains the VP2 protein, and the VP2 protein contains the VP3 protein. The three share a stop codon, in which the extra region of the VP1 protein (abbreviated as VP1U) contains a phospholipase protein, which is responsible for dissolving the endosomal membrane and is hidden inside the viral capsid in a natural state. The VP3 site mutation of the viral capsid affects the ability of the virus to bind to the host cell, while the site mutation in the VP1U region affects the ability of the virus to escape host immunity.

[0036] Since the virus capsid is mainly composed of three tightly bound VP3 proteins, in the early stage of infection, the virus enters the cell through the receptor binding displayed on the surface of VP3 and is wrapped by the cell's endosomes. Under the influence of the intracellular microenvironment, the virus capsid conformation continues to change, and eventually exposes the VP1U phospholipase region hidden inside the capsid, which then dissolves the endosomal membrane to facilitate virus escape and infection. Therefore, the entire capsid protein of the virus determines the strength of the virus's infection ability.

[0037] The present invention obtains a novel goose parvovirus NMG21 strain with reduced toxicity by continuously passage 50 generations of duck embryo fibroblasts to cause feather loss on the back of ducks. The attenuated goose parvovirus attenuated strain NGPV-NMG50 is obtained by passing the clinically isolated novel goose parvovirus NMG21 strain that can cause feather loss on the back of ducks. The homology of the attenuated strain with other NGPV wild strains in GenBank is between 98.5% and 99.6%, and the amino acid homology is between 97.6% and 99.2%, indicating that NGPV-NMG50 comes from the wild strain of NMG21 strain rather than the vaccine strain available on the market.

[0038] Through whole genome sequencing analysis, NGPV-NMG50 and NMG21 wild strains only have two nucleotide site mutations in the VP1 region. The mutations change the coding order of the codons, resulting in differences in the amino acids at the two locations. The two mutations are: the nucleotide at position 233 of the VP1 genome of the wild strain of the new goose parvovirus mutates from G to A, causing the amino acid encoded by this site to change from serine (AGC) to asparagine (AAC), and the nucleotide at position 1700 of the VP1 gene mutates from G to C, causing the amino acid encoded by this site to change from glycine (GGA) to alanine (GCA). The sequenced VP1 genome nucleotide sequence of the NMG21 wild strain is shown in SEQ ID NO: 1, as follows:

[0039] SEQ ID NO:1:

[0040]

[0041] The amino acid sequence of the protein encoded by the VP1 genome of the NMG21 wild-type virus strain is shown in SEQ ID NO:3, and is as follows:

[0042] SEQ ID NO:3:

[0043] .

[0044] The sequenced VP1 genome nucleotide sequence of the attenuated strain of NGPV-NMG50 is shown in SEQ ID NO: 2, and is as follows:

[0045] SEQ ID NO:2:

[0046]

[0047] The amino acid sequence of the protein encoded by the VP1 genome of the NGPV-NMG50 attenuated strain is shown in SEQ ID NO:4, as follows:

[0048] SEQ ID NO:4:

[0049] MSTFLDSFEEWYETAAASWRNLKAGAPQPKPNQQSQSVSPDREPERRDNNRGFVLPGYKYLGPGNGLDKGPPVNKADNVALEHDKAYDLQLKSGDNPYIKFNHADQDFIDSLQDDYSFGGNLGKAVFQAKKRILEPFGLVEEPINTAPAKKNTGKLTDHYPVVKKPKLTEEVSAGGGSSAVQDGGATAEGTEPVAASEMAEGGGGAMGDSSGGADGVGNASGNWHCDSQWMGNTVITKTTRTWVLPSYNNHIYKAITSGTAQDANVQYAGYSTPWGYFDFNRFHCHFSPRDWQRLINNHWGIRPKSLKFKIFNVQVKEVTTQDQTKTIANNLTSTIQVFTDDEHQLPYVLGSATEGTMPPFPSDVYALPQYGYCTMHTNQNGARFNDRSAFYCLEYFPSQMLRTGNNFEFTFDFEEVPFHSMFAHSQDLDRLMNPLVDQYLWNFNEVDSNRNAQFKKAVKGAYGTMGRNWLPGPKFLDQRVRAYSGGADNYANWNIWSNGNKVNLKDRQYLLQPGPVSAAHTEREASSIPAQNSLGIAKDPYRSGSTTAGISDIMVTDEQEVAPTNAVGWKPYGRTVTNEQNTTTAPTSSDLEVLGALPGMVWQNRDIYLQGPIWAKIPKTDGKFHPSPNLGGFGLHNPPPQVFIKNTPVPADPPVEYVHQKWNSYITQYSTGQCTVEMVWELRKENSKRWNPEIQFTSNFSNRTSIMFAPNETGGYVEDRLIGTRYLTQNL。

[0050] The genome sequencing results showed that the NGPV-NMG50 weak strain was a weak strain whose mutation pattern was inconsistent with previous vaccine strains. The mutations at two nucleotide sites changed the coding order of the codons, resulting in differences in the amino acids encoded at the two sites. The virus had a small mutation range, and only two sites showed a significant virulence reduction effect, affecting the virus's infection level. Other GenBank waterfowl parvovirus strains and commercial vaccine strains did not have this mutation, which provided valuable biological samples for studying the virulence gene sites of waterfowl parvovirus.

[0051] Based on this research, the inventors of this application conducted infection experiments by injecting NGPV-NMG50 into susceptible ducklings. The results showed that the ducks injected with NGPV-NMG50 did not experience any local or systemic adverse reactions. During the experiment, the NGPV-NMG50 strain had no virulence reversion and good stability. Furthermore, the minimum lethal dose of the wild strain of NMG21 was screened and it was found that the highest dilution factor of the wild strain of NMG21 that could kill ducklings was 10 4 The dilution multiple is the minimum infection dose of the wild-type NMG21 strain. The optimal immune dose of the weak NGPV-NMG50 strain was screened by the minimum lethal dose of the wild-type NMG21 strain, and the optimal concentration of the weak NGPV-NMG50 strain was determined to be 10 3 The dose is diluted 100 times, which is the lowest dose of weak strain that can protect ducklings from infection with NGPV wild strain.

[0052] In summary, compared with the prior art, the present application mainly has the following outstanding technical effects:

[0053] (1) Through research, a new goose parvovirus NGPV-NMG50 attenuated strain was obtained. Through genome sequencing and comparison analysis of the NMG21 wild strain, two artificial attenuation sites that can weaken the virulence of the new goose parvovirus were identified, laying a solid foundation for the development of a new goose parvovirus vaccine or immune product prepared with the attenuated strain.

[0054] (2) The artificial attenuation sites provided in this application can be used to genetically modify wild strains, and the strains with weakened virulence can be cultured and used to prepare vaccines. This can avoid the risk of strong toxic spread caused by the use of strong strains for culture, improve biosafety, and reduce production costs.

[0055] (3) The live vaccine prepared in this application using NGPV-NMG50 as the immunogen can achieve a 100% protection rate against feather loss in ducks without the addition of adjuvants. The minimum lethal dose of the virus was determined by diluting the wild-type NMG21 strain in multiples, and then the optimal immune dose of the NGPV-NMG50 weak strain was screened, obtaining the optimal immune dose results for the NGPV-NMG50 weak strain in clinical use.

[0056] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0057] The test materials used in the examples and comparative examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out according to conventional test methods or the operating instructions recommended by the supplier.

[0058] The deposit information of the wild strain of NGPV-NMG21 used in this example is as follows:

[0059] Strain name: NGPV-NMG21;

[0060] Classification and naming: New goose parvovirus ( Anser anser novus parvovirus );

[0061] Depository: General Microbiology Center, China National Microbiological Culture Collection Administration;

[0062] Abbreviation of depository institution: CGMCC;

[0063] Address of the depository institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0064] Deposit date: October 24, 2024;

[0065] Deposit number: CGMCC No.46205.

[0066] Example 1: Obtaining a novel goose parvovirus attenuated strain NGPV-NMG50

[0067] The NGPV-NMG21 wild strain with the deposit number of CGMCC No.46205 was obtained by continuous subculture in duck embryo fibroblasts (DEF), as follows:

[0068] The wild strain of NMG21 was propagated through DEF cells for 10 generations, and the time for the appearance of CPE in the cells was not significantly delayed. When the virus was propagated to the 20th generation, the time for the appearance of CPE in the cells extended from 72 hours to 96 hours, and the CPE phenomenon caused by the virus on the cells began to weaken. Starting from the 30th generation, the virus began to no longer cause CPE in the cells, and the cells were continuously observed for more than 120 hours and still no typical parvovirus infection lesions appeared. Cell pellets at different time points after virus infection were collected, and the virus growth curves of NGPV-NMG21 and NGV-NMG50 were determined using the qPCR method ( Figure 1), and found that the virus's replication ability did not decrease significantly. This means that the virus only mutated in the virulence gene during continuous passage, while the gene region that determines the virus's replication ability was conserved.

[0069] In order to explore whether the virulence of the virus would be further weakened or strengthened during the passage process, the NMG21 wild strain was continuously passaged through DEF cells for 50 generations in the same way to obtain a weak strain. The virus completely lost its pathogenicity to DEF cells while maintaining its own replication level. The strain of the 50th passage was named NGPV-NMG50. The replication level of NGPV-NMG50 was identified using an indirect immunofluorescence assay (IFA). The results are as follows: Figure 2 As shown, the CPE phenomenon of NGPV-NMG21 virus is more obvious, the content of cells in the field of view is small (blue represents the cell nucleus), and the virus causes great damage to the morphology of the cells. After the DEF cell membrane is dissolved, the virus is diffusely distributed inside the cells (green fluorescence represents the virus content). The NGPV-NMG50 weak strain cannot cause CPE phenomenon to cells. The cell morphology is intact, the cell membrane is intact, the cells are closely arranged, and the green fluorescence content is low. This result, combined with the results of the virus growth curve, proves that NGPV-NMG50 significantly reduces the lethality to susceptible cells while maintaining the replication level.

[0070] The steps of indirect immunofluorescence assay are as follows:

[0071] (1) Preparation of DEF cells:

[0072] Take 11-day-old Cherry Valley duck embryos, disinfect the eggshells with alcohol or iodine, and remove the eggshells along the edge of the air chamber. Use sterile tweezers to clamp the embryo neck and place it in a sterile glass culture dish, and use scissors to remove the head, limbs and internal organs. Move the embryo to a small beaker, rinse it repeatedly with PBS reagent, and then cut the embryo into chyme. Use a sterile Pasteur pipette to place the chyme embryo in a 50 mL sterile centrifuge tube, add 4 times the volume of EDTA-trypsin, and place it in a 37℃ water bath for 10 min. Add DMEM containing 3% serum to the embryo treated with trypsin, use a Pasteur pipette to blow repeatedly several times to blow away the tissue blocks and disperse the cells evenly. Use 4 layers of gauze to filter the suspension to remove the unprocessed tissue blocks, use a red blood cell counting plate to count the cells, and then divide the cells into 6-well cell culture dishes and culture them at 37℃ and 5% CO2 concentration.

[0073] (2) Indirect immunofluorescence detection

[0074] Immediately after the preparation of DEF cells, the virus was inoculated into the cell fluid to synchronize virus replication with cell replication. After the virus infected DEF for 72 hours, it was washed 3 times with PBS and fixed with paraformaldehyde, incubated with primary antibody at 4°C overnight, and detected with FITC-labeled goat anti-mouse IgG (H+L) fluorescent secondary antibody to observe the lethality of the virus to susceptible cells.

[0075] Example 2: Analysis of biological characteristics of NGPV-NMG50 weak strain

[0076] (1) Virus titer: The prepared duck embryo fibroblasts were used to determine the virus TCID50, as follows:

[0077] DEF cells were prepared according to the method described in Example 1, and the cells were transferred to a 96-well plate for culture. When the cells grew to 90% density, the virus solution diluted tenfold was inoculated into the cells in the 96-well plate, and 8 replicates were set for each dilution, and 200 uL of the virus dilution solution was inoculated into each well. The inoculated cells were placed in a 37°C incubator and cultured for 96 hours. The cells were observed every day for pathological changes, and the number of infected wells and uninfected wells for each dilution was recorded.

[0078] According to the results of duck embryo fibroblast infection, the TCID of the virus was calculated according to the Reed-Muench method. 50 , NGPV-NMG21 wild strain TCID 50 The Reed-Muench method calculation formula is:

[0079] =0.39

[0080] TCID 50 = logarithm of dilution factor above 50% mortality + difference between logarithms of dilution factor × distance ratio = -7.39

[0081] TCID 50 = 10 -7.39 / 0.2 mL

[0082] Table 1: TCID of NGPV-NMG21 wild strain 50 The results of the measurement

[0083]

[0084] According to the results of duck embryo fibroblast infection, the TCID of the virus was calculated according to the Reed-Muench method. 50 , NGPV-NMG50 weak strain TCID 50 The Reed-Muench method calculation formula is:

[0085] =0.61

[0086] TCID 50 = logarithm of dilution factor above 50% mortality + difference between logarithms of dilution factor × distance ratio = -4.61

[0087] TCID 50 = 10 -4.61 / 0.2 mL

[0088] Table 2: TCID of NGPV-NMG50 attenuated strains 50 The results of the measurement

[0089]

[0090] The results showed that the NMG21 wild strain had a higher titer in duck embryo fibroblasts, TCID 50 Reach 10 -7.39 / 0.2mL, TCID of NGPV-NMG50 weak strain 50 Reduce to 10 -4.61 / 0.2 mL.

[0091] (2) Biological information analysis: Whole genome sequencing analysis showed that the NGPV-NMG50 and NMG21 wild strains only had two nucleotide site mutations in the VP1 region (the nucleotide sequence of the VP1 genome of the NMG21 wild strain was shown in SEQ ID NO:1, and the nucleotide sequence of the VP1 genome of the NGPV-NMG50 attenuated strain was shown in SEQ ID NO:2). These two types of sites are not degenerate amino acid bases, so the mutation also led to mutations in two amino acid sites (the amino acid sequence encoded by the VP1 genome of the NMG21 wild strain is shown in SEQ ID NO:3, and the amino acid sequence encoded by the VP1 genome of the NGPV-NMG50 attenuated strain is shown in SEQ ID NO:4).

[0092] The two mutations are as follows: The first mutation is located at the 233rd nucleotide of the VP1 genome. The mutation causes the amino acid encoded by this site to change from AGC (serine) to AAC (asparagine). The change in amino acid polarity affects the phospholipase region of the virus, which makes it difficult for the virus to escape from the lysosome after infecting the cell, ultimately reducing the virus's infection level. Such mutations exist in a few commercial attenuated vaccine strains of waterfowl parvovirus. The other site is a mutation that has never appeared in all current vaccine strains and wild strains. This site is located at the 1700th nucleotide of the VP1 gene. The mutation causes the amino acid encoded by this site to change from GGA (glycine) to GCA (alanine). Protein modeling by AlphaFold3 found that the mutation blocked a receptor binding site on the surface of the viral capsid protein, potentially affecting the affinity between the virus and the host cell, which in turn led to a significant decrease in the virus's ability to infect.

[0093] The homology of nucleotides between NGPV-NMG50 and the commercial attenuated vaccines GD strain (DQ665790) and SYG61-41 strain (DQ299942) in my country is 92.5% and 93.1%, and the homology of amino acids is 94.9% and 95.6%. At the second mutation site of the VP1 gene, no similar mutation has occurred in any commercial attenuated vaccine. Therefore, the NGPV-NMG50 attenuated strain is a attenuated strain whose mutation pattern is inconsistent with that of previous vaccine strains, and the mutation range of the virus is small. Only two sites show a significant virulence reduction effect, which provides a valuable biological sample for studying the virulence gene sites of waterfowl parvovirus.

[0094] (3) Safety: 20 healthy Cherry Valley ducks without maternal antibody interference at 1 day old were selected and divided into an experimental group and a control group, with 10 ducks in each group. In the control group, each Cherry Valley duck was injected intramuscularly with 0.2 mL of NMG21 wild strain stock solution. In the experimental group, each Cherry Valley duck was injected intramuscularly with 0.2 mL of NGPV-NMG50 weak strain stock solution. After the injection, the ducks were observed for 28 days. Within 28 days, the Cherry Valley ducks injected with NMG21 wild strain showed the classic short beak and long tongue characteristics, while the Cherry Valley ducks injected with NGPV-NMG50 weak strain had no infection characteristics and were in good mental state. The results showed that the live vaccine for preventing the new goose parvovirus prepared with NGPV-NMG50 weak strain as the immunogen is safe and will not cause any local and systemic adverse reactions in the ducks.

[0095] Example 3: Immune protection and stability testing of live vaccines prepared from NGPV-NMG50 attenuated strain

[0096] (1) Protection against short-beaked dwarfism syndrome:

[0097] 90 one-day-old ducklings (Cherry Valley ducks) without NGPV maternal antibodies were selected and divided equally into a protection group, a test group, and a control group, with 30 ducks in each group. The treatment of each group is as follows:

[0098] Protection group: Each duckling was intramuscularly injected with 0.2 mL 1000TCID 50 NGPV-NMG50 weak strain, 5 days after injection, each duckling was injected intramuscularly with 0.2 mL 1000TCID 50 NMG21 virulent strain;

[0099] Experimental group: Each duckling was intramuscularly injected with 0.2 mL PBS. Five days after the injection, each duckling was intramuscularly injected with 0.2 mL 1000 TCID 50 NMG21 virulent strain;

[0100] Control group: Each duckling was intramuscularly injected with 0.2 mL PBS. Five days after the injection, each duckling was intramuscularly injected with 0.2 mL PBS again.

[0101] After the two injections, the ducks were observed for 28 days. The results showed that the ducks in the protection group did not show the typical pathological characteristics of short beaks, long tongues, and feather loss, and no deaths occurred. The mortality rate of the experimental group reached 36%, and the proportion of short beaks, long tongues, and feather loss among the groups reached more than 90%. The mental state of the control group was good, and no deaths occurred during the observation period. The results show that the protection rate of the weak strain of NGPV-NMG50 for ducklings reached 100%, which can effectively establish an immune barrier for ducklings against the new goose parvovirus.

[0102] (2) Stability test of NGPV-NMG50 weak strain:

[0103] The attenuated strain NGPV-NMG50 stock solution was injected into 1-day-old susceptible ducklings (Cherry Valley ducks), 1mL / bird. The virus can be detected and isolated in the liver of infected ducks, and the isolated virus is used to continue to infect 1-day-old ducklings. After the virus is isolated, the animal infection test is continued using the same method, for a total of 6 generations. During the entire observation process, there were no deaths in the ducklings, and there was no short beak, long tongue, or feather loss in the later stage. Therefore, NGPV-NMG50 has no virulence reversion phenomenon, and the attenuated strain has good stability.

[0104] Example 4: Screening for the optimal immunization dose for preparing live vaccines using the NGPV-NMG50 attenuated strain

[0105] The method for screening the optimal immune dose for preparing live vaccine from NGPV-NMG50 attenuated strain includes: firstly diluting the NMG21 wild strain in multiples to determine the minimum lethal dose of the virus, and then screening the optimal immune dose of NGPV-NMG50 attenuated strain using the minimum lethal dose of the wild strain, in order to obtain the optimal immune dose of NGPV-NMG50 attenuated strain in clinical use.

[0106] 1. Screening of the minimum lethal dose of NMG21 wild strain

[0107] One hundred one-day-old Cherry Valley ducks without NGPV maternal antibody interference were randomly divided into 10 groups, including 9 experimental groups and one control group, with 10 ducks in each group. The NMG21 wild strain was diluted in a 10-fold concentration gradient to 10 1 -10 9 There were 9 gradients in total. Each duckling in the 9 test groups was inoculated with 0.2 mL of NMG21 strong strain at different dilutions by intramuscular injection, and each duckling in the control group was injected with 0.2 mL of PBS intramuscularly. The groups were isolated and raised, and the infection was observed for 28 days. The infected ducks were dissected and the morbidity was recorded.

[0108] The test results showed that the wild strain of NMG21 was diluted 10 5 Cherry Valley ducks were infected with 10% and above concentration gradients. No deaths occurred in the ducks. They only showed depression and slight feather loss. No individuals with short beaks and long tongues appeared. Only slight swelling of the bursa of Fabricius was found in autopsy. 4 Infection of Cherry Valley ducks with concentrations below 100% resulted in serious morbidity, with a high mortality rate in the group. Most of the individuals had short beaks and long tongues, and had severe feather loss. The results are shown in Table 3.

[0109] Table 3: Screening of the minimum lethal dose of NMG21 wild strain

[0110]

[0111] Note: The individuals with short beaks, long tongues and shed feathers are the number of Cherry Valley ducks surviving infection / the total number of Cherry Valley ducks.

[0112] As can be seen from Table 1, the highest dilution factor at which the wild strain of NMG21 can kill ducklings is 10 4 The dilution multiple of NMG21 wild strain is the minimum infection dose.

[0113] 2. Screening of the minimum immune dose of NGPV-NMG50 attenuated strain

[0114] One hundred one-day-old Cherry Valley ducks without NGPV maternal antibody interference were randomly divided into 10 groups, 9 experimental groups and 1 control group, with 10 ducks in each group. NGPV-NMG50 was diluted in 10-fold concentrations, with a total of 9 dilutions. Each duckling in the 9 experimental groups was inoculated with 0 1 -10 9 Different dilutions of NGPV-NMG50 attenuated strains were injected 0.2 mL at 3 days after injection. Each duckling in the 9 test groups was injected intramuscularly with 0.2 mL 10 -4 Each duckling in the control group was intramuscularly injected with 0.2 mL PBS. Three days after the injection, each duckling in the control group was intramuscularly injected with 0.2 mL 10 - 4 The above groups were isolated and kept for 28 days after infection, and the disease status of infected ducks was recorded by autopsy.

[0115] The test results showed that the control group died after being infected with the wild strain of NMG21, and the proportion of short beaks, long tongues and feather loss was high. 3 The weak strain of NGPV-NMG50 with a dilution of 10 or less can completely protect the infected group. The ducks are in good spirits, with no death or typical parvovirus lesions. 4 Strains with multiples of 5 or more could not provide effective protection against wild-type NGPV strains, and the ducks showed typical NGPV pathological characteristics of varying severity. The results are shown in Table 4.

[0116] Table 4: Screening of the minimum immune dose of NGPV-NMG50 attenuated strain

[0117]

[0118] Note: The individuals with short beaks, long tongues and shed feathers are the number of Cherry Valley ducks surviving infection / the total number of Cherry Valley ducks.

[0119] As can be seen from Table 2, the optimal concentration of NGPV-NMG50 weak strain is 10 3 The dose is diluted 100 times, which is the lowest dose of weak strain that can protect ducklings from infection with NGPV wild strain.

[0120] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification made within the spirit and principle of the present application shall be deemed to be appropriate.

[0121] Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. A novel attenuated strain of goose parvovirus, characterized in that: The attenuated strain is obtained by subjecting the VP1 protein of the novel wild-type goose parvovirus to site-directed mutagenesis at positions 78 and 567 of the amino acids; The amino acid sequence of the attenuated strain VP1 protein is shown in SEQ ID NO: 4; The new wild strain of goose parvovirus is NGPV-NMG21 strain, which was deposited in the General Microbiology Center of China Culture Collection Administration on October 24, 2024, with the deposit number: CGMCC No.46205.

2. The method for constructing the attenuated strain according to claim 1, characterized in that: The steps include: Through genetic engineering, the 233rd and 1700th nucleotides of the VP1 gene of the new wild-type goose parvovirus strain were subjected to site-directed mutation to obtain a weak strain with reduced virulence. The novel wild-type goose parvovirus strain is the NGPV-NMG21 strain; The site-directed mutagenesis specifically involves: mutating the nucleotide at position 233 of the VP1 genome of the novel wild-type goose parvovirus from G to A and mutating the nucleotide at position 1700 from G to C; The VP1 genome nucleotide sequence of the attenuated strain obtained after the site-directed mutagenesis is shown in SEQ ID NO:

2.

3. Use of the attenuated strain according to claim 1 in the preparation of a vaccine for preventing or treating a novel goose parvovirus.

4. The use according to claim 3, characterized in that: The vaccine is a live attenuated vaccine.

5. The use according to claim 3, characterized in that: The novel goose parvovirus vaccine is prepared by the following steps: The weak strain of the novel goose parvovirus is inoculated into duck embryo fibroblast DEF for culture, and the culture fluid containing the immune effective amount is used as an immunogen to prepare a vaccine for preventing or treating the novel goose parvovirus.

6. The use according to claim 3, characterized in that: The vaccine may further include a pharmaceutically acceptable adjuvant.

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